Analogs of alpha galactosylceramide and uses thereof

ABSTRACT

Compounds of formula 
     
       
         
         
             
             
         
       
         
         
           
             wherein the variable are defined in the specification, are used in compositions which stimulate T cell responses.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/083,673filed May 21, 2009, which is a §371 of PCT/US2006/041592 filed Oct. 25,2006, now U.S. Pat. No. 8,039,670 and claims priority from U.S.Provisional Patent Applications Nos. 60/730,171 filed Oct. 25, 2005;60/756,505 filed Jan. 5, 2006; and 60/761,110 filed Jan. 23, 2006.

FIELD OF THE INVENTION

This invention relates to new ceramide derivatives and their synthesis.These compounds are efficacious as immune stimulators, and have aneffect that may be referred to as an immunomodulatory or adjuvanteffect.

BACKGROUND OF THE INVENTION

α galactosylceramide, compound A, and its derivatives, have been knownas biologically active agents for some time. See, e.g., U.S. Pat. No.5,936,076 to Higa, et al., and U.S. Pat. No. 6,531,453 to Taniguchi, etal., describing several derivatives as anti-tumor agents as well asimmunostimulators, both of these being incorporated by reference intheir entirety.

The base compound, i.e., α-galactosylceramide or “αGal-Cer” hereafter isdescribed by Nattori, et al., Tetrahedron, 50:2271 (1994), incorporatedby reference, has itself been shown to inhibit tumor growth. See,Koejuka, et al., Recent Res. Cancer, 1:341 (1999). Sharif, et al.,Nature Med., 7:1057 (2001), and Hong, et al., Nature Med., 7:1052(2002), show efficacy against type I diabetes.

Study of the structure of αGal-Cer shows that it contains a sphingosinechain. Truncation of this chain has been shown, by Miyamoto, et al.,Nature, 413:531 (2001), to result in a compound preventing autoimmuneencephalomyelitis.

In parallel work it has been shown that natural killer T cells (NKTcells) recognize lipid antigens that are presented by the majorhistocompatibility complex-class I like protein, CD1d, for example. See,Godfrey et al., J. Clin. Invest., 114:1379-1388 (2004).

Singh, et al., J. Immunol., 163:2373 (1999), and Burdin, et al., Eur. J.Immunol., 29:2014 (1999), have shown that αGal-Cer and CD1d potentiateTh2-mediated, adaptive immune responses, via activation of Vα14 naturalkiller T (NKT) cells.

The proposed mechanism by which αGal-Cer prevents disease is its abilityto suppress interferon-gamma, but not interleukin-4, by NKT cells. See,e.g., Brossay, et al., J. Exp. Med., 188:1521 (1998); Spada, et al., J.Exp. Med., 188:1529 (1998), who showed the recognition of αGal-Cer byNKT cells, suggesting therapeutic efficacy in humans.

αGal-Cer has been developed as a potential therapeutic compound andtaken into clinical testing, see, for example, Giaccone et al., ClinCanc. Res., 8, 3702-3709 (2002). However, following treatment withαGal-Cer, the level of NKT cells in the peripheral blood of treatedcancer patients treated fell to undetectable levels within 24 hours oftreatment and failed to regain pretreatment levels within the remainingtime course of the study.

Loss of circulating levels of NKT cells could represent a significantlimitation therapeutically as it could suggest that therapeuticstimulation of NKT cells could not be used as a repeated treatment.

There is thus an interest in synthesis of analogues of αGal-Cer whichact as stimulators of NKT cells but which do not lead to rapid loss ofcirculating levels of NKT cell populations after therapeuticadministration.

Various publications describe synthesis of αGal-Cer and its derivatives.An exemplary, but by no means exhaustive list of such referencesincludes Morita, et al., J. Med. Chem., 38:2176 (1995); Sakai, at al.,J. Med. Chem., 38:1836 (1995); Morita, et al., Bioorg. Med. Chem. Lett.,5:699 (1995); Takakawa, et al., Tetrahedron, 54:3150 (1998); Sakai, atal., Org. Lett., 1:359 (1998); Figueroa-Perez, et al., Carbohydr. Res.,328:95 (2000); Plettenburg, at al., J. Org. Chem., 67:4559 (2002); Yang,at al., Angew. Chem., 116:3906 (2004); Yang, at al., Angew. Chem. Int.Ed., 43:3818 (2004); and, Yu, et al., Proc. Natl. Acad. Sci. USA,102(9):3383-3388 (2005).

Studies have been conducted to examine the biological impact of theαGal-Cer molecule, when modifications of its structure were made. Higa,et al., supra, as well as, Zhou, at al., Org. Lett., 4:1267 (2002);Schmieg, et al., J. Exp. Med., 198:1631 (2003), Barbieri, a al., J. Org.Chem., 468 (2004); and Fan, et al., Tetrahedron, 61:1855 (2005), areexamples of the limited literature on this topic. Tsuji, et al., J. Exp.Med., 198:1631 (2003), prepared a synthetic, C-glycoside analogue, i.e.,α-C-Gal-Cer, which acts on NKT cells, in vivo, stimulating enhanced,Th1-type immune responses in mice. The protection against microbialinfection and anti-tumor efficacy (Sköld, at al., Infect. Immun.,71:5447 (2003); Sharif, et al., supra; Hong, at al., supra) are ofspecial interest.

Additional work on the mechanism of action of these compounds is shownby, for example, Parekh, et al., J. Immunol., 173:3693-3706 (2004), andBrossay, et al., supra.

Examples of US patents and patent applications or International patentapplications describing instances of such derivatives and or thebiological activity of αGal-Cer analogs include U.S. Pat. No. 5,936,076to Higa, et al., and U.S. Pat. No. 6,531,453 to Taniguchi, et al., U.S.Pat. No. 5,853,737 to Modlin et al., US Patent Application 2003030611 toJiang et al., US Patent Application 20030157135 to Tsuiji et al., USPatent Application 20040242499 to Uematsu et al and International PatentApplications describing No. PCT/JP20021008280 to Yamamura et al.

Essentially all of these prior examples describe analog structures basedon αGal-Cer. Identification and characterization of molecules which arenot glycolipids, such as αGal-Cer and its analogs, has been limited.Examples of patent applications describing structures which do notappear to be analogous to αGal-Cer include acyl peptides of US PatentApplication No. 20040265976 to Moody et al., and JP Patent Application34540997 to Masunaga et al.

We have now found a novel group of compounds that substantially mimicthe binding properties of α-GalCer with the human CD1d molecule, butdiffers significantly in the interaction with T-cell receptors (TCR),leading to unexpected and advantageous properties compared to α-GalCer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of dissociation rate of OCH and α-GalCer.Dissociation of ligand from hCD1d. The indicated hCD1d (α-Gal Cer orOCH) complex was loaded onto a Surface Plasmon resonance (Biacore)sensor surface at t=0 and the amount of hCD1d remaining at the indicatedtime point measured using the Fab 9B.

FIG. 2 Comparison of TCR Binding Affinity for OCH and α-GalCer;

Affinity and kinetics of iNKT TCR binding to hCD1d complexed withα-GalCer or OCH. Increasing concentrations from 0.4 μM to 194 μM(two-fold dilution) of iNKT TCR were injected for 5 seconds over theindicated hCD1d-GSL complexes. The binding responses of 5 concentrationsare shown superimposed. The panels on the right show binding response atequilibrium.

FIG. 3 Surface Plasmon Resonance (Biacore) Measurements of Low AffinityAnalogs of α-GalCer

The affinities of a soluble NKT cell TCR for human CD1d moleculesrefolded with different analogs of α-GalCer were measured. Equilibriumbinding and kinetic measurements respectively were made for α-GalCer (Aand B), threitolceramide (C and D), 4S-threitolceramide (E and F),4R-threitolceramide (G and H). The K_(d), values (μM) were calculatedfrom equilibrium binding. The K_(on) values shown were calculated fromK_(off) and K_(d).

FIG. 4 Human DC are Matured with Analogs of α-GalCer In Vitro whenCo-Cultured with iNKT Cells

Different analogs of α-GalCer as indicated or LPS were added toco-cultures of human DC and iNKT cells. (A) Maturation of the DC wasassessed after 40 h, gating on CD11c positive cells. Histograms indicatethe CD83 and CD86 profiles of DC from different treatments; thepercentages indicate the percentage of CD83hi mature DC and the numbersin parenthesis, the mean fluorescence intensity of the populationcontained within the labeled gate. DC and iNKT cells were co-cultured inthe presence of different analogs of α-GalCer for 24 hours. Supernatantswere analyzed using ELISA for the presence of (B) IL-12p40 released bythe DC and (C) IFN-γ from the iNKT cells. All the compounds testedinduced weaker but significant cytokine production than α-GalCer, withthe exception of arabinitolceramide. (D) Titration of α-GalCer, OCH orthreitolceramide on DCs and co-culture with iNKT cells induces DCmaturation in vitro. Cells were co-cultured in the presence of theindicated concentration of analog and the percentages of DC withincreased CD83/CD80 are shown. (E) Titration of α-GalCer inducessignificant death of DC in vitro when co-cultured with iNKT cells whilelow affinity analogs do not. Viability of α-GalCer or analog-pulsed DCwas assessed after 40 h co-culture in the presence of iNKT cells by flowcytometry. Cells were stained with propidium iodide and the percentageof live CD11c gated cells remaining in the culture are shown.

FIG. 5 Non-glycolipid analogs of α-GalCer stimulate activation of iNKTcells and subsequent DC maturation in vivo. C57BL/6 or iNKT^(−/−) micewere injected i.v. with 1 μg of vehicle, α-GalCer, analog or 25 μg MPL.Twenty hours after injection splenocytes were stained with antibodiesagainst CD11c, B220 and CD86 and analyzed by flow cytometry. (A)Maturation was assessed by the upregulation of CD86 at the cell surface,gating on DC (CD11c⁺). Mean fluorescence intensity for each histogram isindicated. These are representative profiles of two independentexperiments. 2, 6 and 18-24 h after injection as described, the micewere bled and the serum tested with ELISA for the presence of (B) IL-4and (C) IFN-γ released in response to the analog.

FIG. 6 Non-Glycolipid Antigens as Effective Adjuvants whenCo-Administered with a Target Antigen.

(A) C57BL/6 mice were co-injected with 1 μg α-GalCer or analog and 800μg OVA. 6 days later blood samples taken from the tail vein were staineddirectly ex vivo with fluorescent SIINFEKL-K^(b) tetramers and ananti-CD8 antibody and analyzed by flow cytometry. Data are shown astetramer positive cells as a percentage of CD8⁺ cells. (B) 7 days afterinjection, the mice were challenged in a prophylactic setting with 1×10⁶E.G7-OVA tumour cells s.c. and the size of the tumour monitored oversubsequent days. There was little or no tumour growth in mice injectedwith α-GalCer, arabinitol or threitolceramide with OVA.

FIG. 7 Non-Glycolipid Analogs of α-GalCer Stimulate B Cell Maturation inthe Presence of iNKT Cells In Vivo and Subsequent Antibody Production.

(A) C57BL/6 or iNKT^(−/−) mice were injected i.v. with 1 μg of vehicle,α-GalCer, analog or 25 μg MPL. Twenty hours after injection splenocyteswere stained with antibodies against B220 and CD86 and analyzed by flowcytometry. Maturation was assessed by the upregulation of CD86 at thecell surface, gating on B cells (B220⁺). Mean fluorescence intensity foreach histogram is indicated. (B) Simultaneous administration of 1 μgα-GalCer or analogs and 400 μg OVA induces significant OVA-specificIgGs. Mice were bled 11-14 d after administration and the serum testedby ELISA for antibodies. Briefly, ELISA plates were coated with 10 μg/mlOVA and then serial dilutions of sera added and incubated overnight at4° C. before detecting with an HRP-conjugated sheep anti-mouse IgG.

FIG. 8 Expansion of Melan A-specific CD8⁺ T cells with DC pulsed withthreitolceramide is at least as effective as with DC pulsed withα-GalCer in vitro Expansion of Melan A-specific CD8⁺ T cells with DCpulsed with threitolceramide is at least as effective as with DC pulsedwith α-GalCer in vitro. Addition of different α-GalCer analogs andMelan-A₂₆₋₃₅ peptide to human PBMCs induced DC maturation and subsequentexpansion of Melan-A specific CD8⁺ T cells as assessed byHLA-A2/Melan-A₂₆₋₃₅ tetramer analysis. Flow cytometry of day 10-15cultures of PBMCs co-cultured with autologous irradiated APCs, pulsedwith Melan-A₂₆₋₃₅ peptide were stained with the tetramer andanti-CD8⁺-FITC. Percentages of tetramer cells as a percentage of totalCD8⁺ cells±SE are indicated.

FIG. 9 Inositol-based α-GalCer derivatives are functional in vitro.Human monocyte-derived DC were pulsed with 150 ng of different inositolanalogs and co-cultured with iNKT cells for 24 h. The 6-Deoxy- and6-Sulfono-myo-inositolceramide analogs were only slightly less potentthan α-GalCer as assessed by CD83 and CD86 upregulation. Numbersindicate the mean fluorescence intensity of the histogram gated onCD11c⁺ cells. 6-Deoxy and 6-Sulfono-myo-inositolceramide inducesignificant (B) IL-12p40 and (C) IFN-γ production by DC and iNKT cellsrespectively in the supernatants of co-cultured DC and iNKT cells.Supernatants were tested by ELISA and reveal that6-Deoxy-myo-inositolceramide induces release of similar concentrationsof IL-12p40 and IFN-γ to those observed with α-GalCer. Release of (D)IFN-g and (E) IL-4 from iNKT cells after 24 h in culture with C1R-humanCD1d cells pulsed with α-GalCer, 6-Deoxy- or6-Sulfono-myo-inositolceramide. Titrations of the analogs suggest thatthe human iNKT cell TCR has a lower affinity for these compounds.

SUMMARY OF THE INVENTION

According to the invention there is provided a compound of formula I,

in which

-   R¹ represents a hydrophobic moiety adapted to occupy the C′ channel    of human CD1d,-   R² represents a hydrophobic moiety adapted to occupy the A′ channel    of human CD1d, such that R¹ fills at least at least 30% of the    occupied volume of the C′ channel compared to the volume occupied by    the terminal nC₁₄H₂₉ of the sphingosine chain of    α-galactosylceramide when bound to human CD1d and R² fills at least    30% of the occupied volume of the A′ channel compared to the volume    occupied by the terminal nC₂₅H₅₁ of the acyl chain of    α-galactosylceramide when bound to human CD1d-   R³ represents hydrogen or OH,-   R^(a) and R^(b) each represent hydrogen and in addition, when R³    represents hydrogen, R^(a) and-   R^(b) together may form a single bond,-   X represents or —CHA(CHOH)_(n)Y or —P(═O)(O⁻)OCH₂(CHOH)_(m)Y, in    which Y represents CHB₁B₂,-   n represents an integer from 1 to 4, m represents 0 or 1,-   A represents hydrogen,-   one of B₁ and B₂ represents H, OH or phenyl, and the other    represents hydrogen or one of B₁ and B₂ represents hydroxyl and the    other represents phenyl, in addition, when n represents 4, then A    together with one of B₁ and B₂ together forms a single bond and the    other of B₁ and B₂ represents H, OH or OSO₃H and pharmaceutically    acceptable salts thereof.

According to the invention we also provide a process for the productionof compounds of formula I, or a salt thereof, or a pharmaceuticallyacceptable derivative thereof, which comprises removal of one or moreprotecting groups from a corresponding compound of formula II,

in which X^(p) represents protected X, R^(p) is a hydroxyl protectinggroup, R^(3p) represents a protected hydroxyl group or a hydrogen andR¹, R², R^(a) and R^(b) are as defined above.

DETAILED DESCRIPTION OF THE INVENTION

Suitable protecting groups in X^(p) that may be used to protect Xinclude protecting groups well known to the person skilled in the art ofthe synthesis of carbohydrates, in particular for protecting isolatedhydroxyl groups and adjacent hydroxyl groups. Examples of protectinggroups are given in Protecting Groups by P. J. Kocienski (Publisher,Thieme Publishing Group, ISBN 3131356030), the contents of which arehereby incorporated by reference. Further examples are given inCarbohydrate Chemistry by Geert-Ian Boons (Editor, G. J. Boons,Publisher, Springer, ISBN 0751403962), the contents of which are herebyincorporated by reference.

Particular OH protecting groups for X^(p) include bis-O-isopropylidene,bis-O-cyclohexylidene, tert-butyldiphenylsilyl, allyl and benzyloxygroups. The deprotection reaction may be carried out withtrifluoroacetic acid in a methanol:dichloromethane mixture at roomtemperature over several days. The deprotection may involve Pd/C in thepresence of hydrogen gas in methanol or methanol/EtOAc mixture. Wherethe XP group contains an O—P bond, the triethylamine may also be addedto form the ammonium salt.

Suitable protecting groups for R^(p) and R^(3p) include O-benzyl groupsand where R^(p) and R^(3p) together forming a bis-O-isopropylidenegroup.

Compounds of formula II, where OR^(p) and R^(3p) are O-benzyl groups, R²is a C₂₅H₅₁ group, and where X^(p) is represented by formula,

may be prepared by the reduction of compound of the formula IIa, whereX^(p) is

And where OR^(p) and R^(3p), R² groups are defined above and where groupW is phenoxythiocarbonyl ester group. The reaction may be carried outwith tributyltin hydride, AIBN in toluene heated to reflux for 4 hours.

Compounds of formula IIa may be prepared from compound of formula III)where X^(p) is represented by formula,

where OR^(p), R^(3p), R² are defined above and W is a hydrogen. Thereaction may be carried out with phenoxythiocarbonyl chloride, pyridine,DMAP and dichloromethane at room temperature for 30 minutes.

Compounds of formula lib may be prepared from compounds of formula IIcwhere X^(p) is represented by formula,

where OR^(p), R^(3p), R² are defined above and W is All group. Thereaction may be carried out with tris(triphenylphosphine)ruthenium (II)chloride, DBU, 90° C., 30 minutes followed by the addition of 1MHCl/acetone.

Compounds of formula II and IIc may be prepared by reacting compounds offormula III,

in which R² is defined above, with a compound of formula IV,

where X^(p), OR^(p), R^(3p), R¹, R^(a) and R^(b) are defined above, savethat X^(p) is not

the reaction may be carried out with EDC, HOBt, TEA in DMF at 45° C. for24 hours.

Compounds of formula III are available commercially or may be made fromcommercially materials by conventional methods per se.

Compounds of formula IV maybe be prepared by reacting compounds offormula V,

where X^(p), OR^(p), R^(3p), R¹, R^(a) and R^(b) are defined above. Thereaction may be carried out with LiAlH₄ in ether at 0° C. warming toroom temperature over 1 hour.

Compounds of formula V, where OR^(p) and R^(3p) are benzyloxy groups andwhere XP is represented by formula,

can be made by the benzyl protection to compounds of formula Va whereOR^(p) and R^(3p) are defined above, and Xp is represented by the group,

The reaction may be carried out in the presence NaH, BnBr, DMF at roomtemperature for 5 hours.

Compounds if formula Va can be made by the deprotection of compound offormula Vb where OR^(p), R^(3p) are define above and X^(p) isrepresented by group,

The reaction may be carried out in the presence hydrochloric acid in atoluene/ethanol mixture,

Compounds of formula V and Vb, may be prepared by reacting compounds offormula VI,

where OR^(p), R^(3p), R¹, R^(a) and R^(b) are defined above with acompound of formula VII,X^(p)—O-L  VIIand where group X^(p) is defined above, save that group X^(P) is notrepresented by formula

The reaction may be carried out in the presence of sodium hydride inTHF, at 0° C. warming to room temperature overnight.

Compounds of formula VI are available commercially or may be made fromcommercially materials by conventional methods per se.

Compounds of formula VII may be prepared by reacting compounds offormula VIII,XP—OH  VIIIwhere X^(p) is above defined above with triflic anhydride indichloromethane and 2,6-di-tert-butylpyridine.

Compounds of formula VIII are available commercially or may be made fromcommercially materials by conventional methods per se.

Or alternatively compounds of formula VIII can take the form ofcompounds of the formula IX:

Where R³ is hydrogen. Compounds of formula IX may be prepared bydeprotecting compounds of formula X,

by catalytic hydrogenation, where the group R³ is defined above, andprotecting group P is a benzyl group. The reaction may be carried outwith 10% Pd/C, H₂, in MeOH/EtOAc (2:3) overnight.

Compounds of formula X may be prepared from compounds of formula XI,

where P is defined above and R³ is a phenoxythiocarbonyl ester. Thereaction may be carried out with tributyltin hydride, AIBN in tolueneheated to reflux for 4 hours.

Compounds of formula XI may be prepared by preparing from compound offormula XII,

where P is defined above and R³ is a hydroxyl group. The reaction may becarried out with phenoxythiocarbonyl chloride, pyridine, DMAP anddichloromethane at room temperature for 30 minutes.

Compounds of formula XII are available commercially or may be made fromcommercially materials by conventional methods per se.

Or further alternatively, compounds of formula VIII, can take the formof compounds of the formula XII,

Where P² is a hydrogen and P³ is either a benzyl group or an A11 group.Compounds of formula XIII may be prepared by the protection of compoundsof formula XIV with a benzyl group,

Where P² and P³ is defined above. The reaction may be carried out withNan, BnBr in toluene heated to reflux for 10 hrs, followed by separationof isomers.

Compounds of formula XIV are available commercially or may be made fromcommercially materials by conventional methods per se.

Compounds of formula II where R² is C₂₅H₅₁, OR^(p) and R^(3p) togetherform a bis-O-isopropylidene protecting group and X^(p) is represented bythe formula,

can be prepared by reacting compounds of formula XV,

where R² is defined above, with compounds of formula XVI

The reaction may be carried out with tetrazole in dichloromethane atroom temperature of 3 hours as an activator, followed by the addition oftert-BuOOH as an oxidant.

Compounds XV and XVI are available commercially or may be made fromcommercially materials by conventional methods per se.

Compounds of formula III can take the form of compounds of the formulaXVII

Compounds of formula XVII may be prepared from compounds of XVIII wheren and Q are defined above,

The reaction may be carried out in the presence of NaOH (where n=20) andLiOH (where n=17) heat to reflux in methanol for 2 hours followed by theaddition of acid.

Compounds of formula XVIII may be prepared from compounds of XIX,

The reaction may be carried out in the presence ofn-butyltriphenylphosphonium bromide (where n=20), andheptenetriphenylphosphonium bromide (where n=17), with sodiumbis(trimethylsilyl)amide at −78° C. overnight.

Compounds of formula XIX may be prepared from compounds of XX, where nis defined above

The reaction may be carried out in the presence DMP in dichloromethaneat room temperature for 3-4 hours.

Compounds of formula XX may be prepared from compounds of XXI, where nis defined above

The reaction may be carried out in the presence of p-TSA in methanol for3-4 hours.

Compounds of formula XXI may be prepared from compounds of XXII, where nis defined above

The reaction may be carried out in the presence of diazomethane in THFover 4 hours at 0° C. warming to room temperature.

Compounds of formula XXII may be prepared from compounds of XXIII, wherem=9 (where n=20), and m=6 (where n=17)

with a compound of formula XXIV

The reaction may be carried out in the presence of MeMgCl, Li₂CuCl₄ inTHF at −20° C. to room temperature over 16 hours.

Compounds of formula XXIII may be prepared from compounds of XXV, wherem is defined above

The reaction may be carried out in the presence of Mg, THF reflux 4hours.

Compounds of formula XXV may be prepared from compounds of XXVI, where Mis defined above

The reaction may be carried out in the presence of 3,4-dihydro-2H-pyran,PPTS.

Compounds XXIV and XXVI are available commercially or may be made fromcommercially materials by conventional methods per se.

Suitable pharmaceutically acceptable salts of the compounds of formula Isalts with suitable bases. Examples of such salts include alkali metal,e.g., sodium and potassium, and alkaline earth metal, e.g., calcium andmagnesium, salts.

The compound of formula I may be obtained in the form of a salt,conveniently a pharmaceutically acceptable salt.

Where desired, such salts may be converted to the free bases usingconventional methods. Pharmaceutically acceptable salts may be preparedby reacting the compound of formula I with an appropriate acid or basein the presence of a suitable solvent.

The compounds of formula I may exhibit tautomerism, they may alsocontain one or more asymmetric carbon atoms and may therefore exhibitoptical and/or diastereoisomerism. Diastereoisomers may be separatedusing conventional techniques, e.g. chromatography or fractionalcrystallisation. The various optical isomers may be isolated byseparation of a racemic or other mixture of the compounds usingconventional, e.g. fractional crystallisation or HPLC, techniques.Alternatively the desired optical isomers may be made by reaction of theappropriate optically active starting materials under conditions whichwill not cause racemisation. We particularly prefer compounds of formulaI in which the stereochemistry of the hydrophilic moiety is analogous tothat found in the α-galactose moiety of α-GalCer.

The conditions for a substantially full occupation of both the A′ and C′channels, as exhibited by α-GalCer by are described in detail in Koch etal, Nature Immunology, 6(8) 819-826 (2005). In Koch et al., cavitieswere identified as surfaces accessible to water molecules (radius, 1.4Å) but no large probes (radius, 6 Å) with the program VOLUMES (R.Esnouf, University of Oxford, Oxford, UK). The open nature of thepockets at the TCR recognition surface required imposition of aself-consistent definition for the outer limit of the pocket, and onthis basis the authors calculated the pocket volumes for mouse CD1d,CD1a and CD1b as well as human CD1d. Although this resulted in somedifferences in absolute values from those reported before, the samerelative trends were noted. Shape complementarity analysis was madeusing the program SC (http://www.ccp4.ac.uk/ccp4i_main.html). Weparticularly prefer compounds of formula that bind to human CD1d with agood shape complementarity, that is with a S_(c) greater than 0.50, morepreferably greater than 0.55, particularly greater than 0.60.

The 26 carbon acyl chain and the 18 carbon sphingosine chain of α GalCerfit into the A′ and C′ pockets, respectively, with good shapecomplementarity (S_(c) 0.61). The total volume of these cavities (1,400Å³) of these cavities in, the human CD binding groove is essentiallyfilled by the hydrocarbon chains. The acyl chain fits into the A′ pocketby adopting a counterclockwise circular curve as viewed from above thebinding groove, filling the pocket. The sphingosine chain adopts astraighter conformation to fit into the C′ pocket and terminates at theend of the binding groove. Thus it is likely that α-GalCer has themaximum lipid chain lengths that are able to fit into theantigen-binding groove of human CD1d. Accordingly we prefer the lengthof R² (the acyl chain) not to exceed 25 carbon-carbon single bonds inlength and R¹ (the sphingosine chain) not to exceed 13 carbon-carbonsingle bonds in length. From binding studies reported in the literature,it is known that carbon-carbon double bonds may be substituted forseveral of the carbon-carbon single bonds, provided that the hydrophobicmoieties are still able to occupy the conformations necessary forbinding with their respective channels. Some studies have shown forexample that the channels are able to accept relatively bulkyhydrophobic residues, such as phenyl.

We prefer compounds of formula I in which R¹ fills at least 35%, morepreferably at least 60%, yet more preferably at least 80% and especiallyat least 90% of the occupied volume of the C′ channel as hereinbeforedefined.

We prefer compounds of formula I in which R² fills at least 40%, morepreferably at least 50%, yet more preferably at least 60%, particularlyat least 70% and especially at least 80% of the occupied volume of theA′ channel as defined hereinbefore.

From x-ray diffraction studies and modeling experiments, it appears thatwhen the R² group is shorter than the preferred maximum length, theremaining space can be occupied by “spacer” molecules which occurnaturally in the body and are sufficiently available to occupy vacantspaces in the CD1d molecule. Such spacer molecules are lipids and thelike. Because of this compounds of formula I in which R² a great dealsmaller than is needed for maximum occupation of the A′ channel willstill bind well to the CD1d molecule.

Preferably, R² is at least one carbon unit (ie methyl) in length, morepreferably at least 5 carbon units in length and particularly at least 8carbon units in length. We prefer compounds of formula 1 in which R²represents a saturated or unsaturated linear hydrocarbon chaincontaining from 1 to 25, more preferably 5 to 25 and particularly 8 to25 carbon atoms.

It appears that the binding of the sphingosine chain is more dependenton the Proportion of occupation of the C′ channel, than the binding ofthe acyl chain to the A′ channel, in that occupation of this channel byspacer molecules has not, as far as the inventors are aware, beenobserved to date. As such, R¹ is preferably at least 5 carbon-carbonsingle bonds in length, more preferably at least 11 carbon-carbon singlebonds in length and especially 12 or 13 carbon-carbon single bonds inlength.

We prefer compounds of formula I, wherein either or both of R¹ or R²contains one or more double bonds. We particularly prefer thosecompounds in wherein either or both of R¹ or R² contains one, two orthree double bonds. We prefer those compounds in which R² containsdouble bonds.

We prefer those compounds wherein the double bonds are cis (Z).

We prefer those compounds in which X represents CHA(CHOH)_(n)CHB₁B₂.

We prefer compounds of formula I in which X representsCH₂(CHOH)_(n)CHB₁B₂.

We prefer compounds of formula I in which n represents 1, 2 or 3,especially 2.

We prefer compounds of formula I and in which n represents 2 which is inthe threo, as opposed to erytho configuration.

We prefer compounds of formula I in which R³ represents hydrogen.

We prefer compounds of formula I in which R^(a) and R^(b) both representhydrogen.

We prefer compounds of formula I in which one of B₁ and B₂ representshydrogen and the other represents hydroxyl.

We prefer compounds of formula I in which m represents 1

We prefer compounds of formula I in which Y represents CH₂OH, CH₂PH orC(OH)(Ph). We especially prefer compounds where Y represents CH₂OH.

We also prefer compounds of formula I in which n represents 4, and whereA and B¹ and B² together form a single bond and the other of B¹ and B²is OH or OSO₃H.

The ability of the compounds of formula I to modulate antigen specificimmune responses enables the compounds to be useful in cancer therapy,preventive and therapeutic vaccines, allergies and autoimmune diseases.

According to the invention there is also provided a compound of formulaI for use as a medicament.

According to the invention there is also provided a method of protectinga mammalian subject against, or treating, a virus, microbial infection,parasite, an autoimmune disease, cancer, allergy or asthma whichcomprises administering to the subject a pharmaceutically effectiveamount of a compound according to the invention which has pharmaceuticalactivity against, or in treating, such a virus, microbial infection,parasite, an autoimmune disease, cancer, allergy or asthma.

According to the invention we also provide the use of compounds offormula I and salts thereof in the preparation of a medicament for thetreatment or prophylaxis of a virus, microbial infection, parasite, anautoimmune disease, cancer, allergy or asthma.

In particular, the compounds of formula I may be used in the treatmentor prophylaxis of the following diseases:

Cancers: For example Basal Cell Carcinoma, Breast Cancer

-   -   Leukemia, Burkitt's Lymphoma, Colon Cancer, Esophageal Cancer,        Bladder Cancer, Gastric Cancer, Head and Neck Cancer,        Hepatocellular Cancer, Hodgkin's Lymphoma, Hairy Cell Leukemia    -   Wilms' Tumor, Thyroid Cancer, Thymoma and Thymic Carcinoma,        Testicular Cancer, T-Cell Lymphoma, Prostate Cancer, Non-Small        Cell Lung Cancer, Liver Cancer, Renal Cell Cancer, and Melanoma.        Viral infections that may be mentioned include:

Viral Hepatitis for example HBV, HCV;

Herpes virus infection for example Herpes simplex virus.

Other skin tropic viruses like human papiloma virus.

Lung tropic viruses like influenza virus or respiratory syncytial virus.

Chronic or acute viral infections with HIV, EBV or CMV or combinationsof viral infections or viral and bacterial infections.

Bacterial infections of the lung with for example Haemophilus influenzaeor mycobacteria for example Mycobacterium tuberculosis and bacterialinfections of the gut with for example helicobacter pylori or the skinlike Staphylococcus aureus.

Asthma, allergen induced asthma, contact dermatitis, psoriasis, Crohn'sdisease.

More especially diseases that may be treated with compounds of formula Iare virus, microbial infection, parasite, cancer.

The compounds of formula I may be used alone or in combination withother therapeutic agents. Combinations with other therapeutic agentsinclude:

Immune modulators like anti CD40/CD40L antibody, anti-CTLA-4 blockingantibody or soluble LAG3 based immune modulators, Toll-like receptoragonists like MPL, CpG, Single-Stranded RNA, nucleotides, nucleotideanalogues like CL087 or loxoribine, polyinosine-polycytidylic acid,flagellin, resiquimod or immiquimod, gardiquimod among others. NODLigands like Muramyl dipeptide, Murabutide or Peptidoglycan,Muramyldipeptide among others, Anti-virals like oseltamivir phosphate,antifungals like Amphotericin B and antibiotics. Antiviral antibodieslike palivizumab.

Other useful combinations include other cancer immune therapeutics likeherceptin, alemtuzumab, gemtuzumab, rituximab, ibritumomab tiuxetan andother monoclonal antibody based cancer treatments. Chemotherapy agents,kinase inhibitors like Imatinib or Erlotinib or cytotoxic agents likecyclophosphamide. Anti-asthmatics and antihistamines andanti-inflammatory drugs could also be used in combination. Otherpotential combinations include vaccine adjuvants like virus-likeparticles (VIPs), liposomes, and artificial antigen presenting cells. Itcan be used as an additive in live cell therapy for example DC-basedimmunotherapy.

Other potential combinations include, cytokine or chemokine blockingantibodies like infliximab, Adalimumab and basiliximab.

We also provide a pharmaceutical composition comprising a compoundaccording to the invention in admixture with a pharmaceuticallyacceptable excipient, carrier or adjuvant. Such formulations aregenerally well known to the person skilled in the art and may beanalogous to those described in EP 0 609 437B, EP-A-1 437 358 and WO2004/028475, the contents of which are herein incorporated by reference.

Compositions in a form suitable for topical administration to the lunginclude aerosols, e.g. pressurised or non-pressurised powdercompositions;

compositions in a form suitable for oesophageal administration includetablets, capsules and dragees;

compositions in a form suitable for administration to the skin includecreams, e.g. oil-in-water emulsions or water-in-oil emulsions;

compositions in a form suitable for administration intravenously includeinjections and infusions; and compositions in a form suitable foradministration to the eye include drops and ointments.

According to the invention there is also provided a pharmaceuticalcomposition comprising, preferably less than 80% and more preferablyless than 50% by weight of a compound of formula I. or apharmaceutically acceptable derivative thereof, in admixture with apharmaceutically acceptable diluent or carrier.

Examples of such diluents and carriers are:

for tablets and dragees—lactose, starch, talc, stearic acid;

for capsules—tartaric acid or lactose; and

for injectable solutions—water, alcohols, glycerin, vegetable oils.

When the compound of formula I is to be administered to the lung it maybe inhaled as a powder which may be pressurised or non-pressurised.Pressurised powder compositions of the compounds of formula may containa liquified gas propellant or a compressed gas. In non-pressurisedpowder compositions the active ingredient in finely divided form may beused in admixture with a larger-sized pharmaceutically acceptablecarrier comprising particles of up to, for example, 100 μm in diameter.

Suitable inert carriers include, e.g. crystalline lactose.

For the above mentioned uses the doses administered will, of course,vary with compound employed, the mode of administration and thetreatment desired. However, in general, satisfactory results areobtained when the compound of formula I is administered at a dailydosage of from about 1 μg to about 20 mg per kg of animal body weight,preferably given in divided doses 1 to 4 times a day or in sustainedrelease form.

For man the total daily dose is in the range of from 70 μg to 1,400 mgand unit dosage forms suitable for administration comprise from 20 mg to1,400 mg of the compound admixed with a solid or liquid pharmaceuticaldiluent or carrier.

The compounds of formula I have the advantage that they are less toxic,more efficacious, are longer acting, have a broader range of activity,are more potent, produce fewer side effects, are more easily absorbed orhave other useful pharmacological properties, than compounds of similarstructure, for example GalCer.

Several of the analogs of the current invention exhibited excellentimmunomodulatory activity as evidenced by the fact that (a) analogsacted as immunostimulants or adjuvants when injected into mice togetherwith antigens as evidenced by the generation of antigen-specific IgGresponses, or antigen-specific cytotoxic CD8+ T lymphocytes responses,or as evidenced by the protection of mice from death when analogs wereinjected as monotherapy into animals in both protective and establishedmodels of tumour growth.

Additionally, analogs acted as immunostimulants when injected asmonotherapy into mice, as evidenced by the induction of IL-12 responses,or as evidenced by the protection of mice from weight loss and/or deathin established models of influenza infection.

Additionally, analogs acted as immunostimulants or adjuvants when addedto samples of human peripheral blood lymphocytes (‘PBLs’) as evidencedby the induction of markers of dendritic cell maturation in such PBLsamples, or when added to samples of human PBLs which were pulsed by theaddition of the known immunogenic MelanA26-35 peptide, ELAGIGILTV (SEQID NO: 1), as evidenced by the induction of CD8+ T cells which werespecific for the target MelanA26-35 peptide.

Importantly, many αGal-Cer analogs of the prior art induce such strongNKT cytokine responses in vivo that NKT cells are driven into anunresponsive state and become refractory to further immunostimulation,see, Parekh et al., J. Clin. Invest., 115:2572-2583 (2005).

In contrast to compounds of the prior art, certain analogs of thecurrent invention are highly effective inducers of human dendritic cellmaturation whilst producing only limited NKT cytokine responses in humanNKT cells compared with αGal-Cer. Such separation of dendritic cellstimulation and consequent maturation in the absence of NKT cytokinesecretion is a unique aspect of the current invention.

This unique and novel aspect is anticipated to allow repeatedstimulation of dendritic cells without exhaustion or depletion of theNKT cell population following administration of compounds of the currentinvention.

In the examples which follow, new molecules are described, together withmethods for their synthesis. These examples are followed by a showing ofthe immunomodulatory properties of these molecules.

The stimulation referred to supra can be seen to provide protection fromconditions in which it is desirable for the immune system to respondeffectively such as infectious disease or cancer.

Additionally, compounds of the invention can be used in combination withTLR ligands such as poly I:C (TLR3), MPL (TLR4), imiquimod (TLR7), 8848(TLR8) or CpG (TLR9) to produce an enhanced immune stimulation andresulting protection from conditions in which it is desirable for theimmune system to respond effectively such as infectious disease orcancer.

Compounds of the invention can also be used as immunostimulants oradjuvants in combined use with antigen materials such as, withoutlimitation, proteins, peptides, or nucleic acids and so forth in orderto produce a protective immune responses, such as a B-cell and IgGantibody response to the administered antigen.

Compounds of the invention can also be used as immunostimulants oradjuvants in combined use with antigen materials such as, withoutlimitation, proteins, peptides, or nucleic acids and so forth in orderto produce a protective immune responses, such as a T-cell or CTLresponse to the administered antigen.

Such antigen materials could be any materials suitable for prevention ortherapy of that particular disease. Specifically, with regards tocancer, examples of tumor associated peptide and protein antigens thatcan be administered to induce or enhance an immune response are derivedfrom tumor associated genes and encoded proteins including MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-AG, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, BADE-1, RAGE-1, LB33/MUM-1, PRAMS, NAG,MAGE-1×2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase,brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1,LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7.For example, antigenic peptides characteristic of tumors include thoselisted in published PCT application WO00/20581 (PCT/US99/21230).

The compounds of the invention are efficacious both in vitro and invivo, and in both mice and humans as has been shown. Hence, one aspectof the invention relates to stimulating an immune response in a subject,by administering one or more of the compounds of the invention with orwithout an antigenic molecule, in an amount sufficient to stimulate afavorable immunologic response in such subject.

It will be clear as well that compositions and or kits, comprising oneor more of the derivatives of the invention, together with one or moreimmunogenic proteins or peptides (as compositions), or as separateportions of derivative and protein or peptide, (as kits), are anotherfeature of the invention.

Other facets of the invention will be clear to the skilled artisan andneed not be reiterated here.

There are two major parameters that determine the activation of NKTcells by its ligand CD1d,

1) The affinity of the iNKT TCR to CD1d (largely determined by thenature of the CD1d bound ligand).

2) The stability of the CD1d complex (determined by the nature of theCD1d bound ligand).

Combined structural, kinetic and functional analyses of soluble TCRbinding to CD1d-lipid complexes and activation of invariant NKT (iNKT)cells have provided important insights into the identification ofoptimal iNKT cell agonists for clinical use. The aim of these studieswas to identify iNKT cell agonists that, unlike α-GalactosylCeramide(α-GalCer), were capable of fulfilling 3 criteria: a) capable ofinducing iNKT cell activation, without over-stimulating iNKT cells tominimise iNKT cell dependent Dendritic Cells (DC) lysis and cytokinestorm; b) capable of ensuring DC maturation c) capable of ensuringoptimal antigen specific T cell priming.

The knowledge derived from the structure of CD1d-α-GalCer specific TCRs(Gadola, Koch et al, J Exp Med, 2006, 203; 699-710) and from thestructure of empty and α-GalCer loaded human CD1d molecules (Koch,Strange et al, Nat Immunol, 2005, 6; 819-26) prompted us to carry out aseries of kinetic and functional experiments to assess the role of thepolar head and the length and saturation of α-GalCer alkyl chains incontrolling the rate of dissociation of lipids bound to CD1d moleculesand the affinity of binding of lipid specific TCR.

To address these questions, we engineered two reagents: i) a soluble TCRfrom an iNKT cell clone and ii) an antibody specific for CD1d-α-GalCercomplex. Using these two reagents we carried out combined kinetic andfunctional studies to compare affinity of iNKT TCR binding to human CD1dmolecules loaded with either α-GalCer or its analogues with eithertruncated acyl and sphingosine chains or modified polar head.

Role of Lipid Length in Controlling Stability of CD1d/Lipid ComplexesiNKT and in Modulating TCR Binding Affinity to CD1d-Lipid Complex.

We refolded in vitro reduced and biotinylated CD1d monomers and loadedthem with a range of α-GalCer analogues with truncated acyl orsphingosine chains. We then analyzed independently the rate ofdissociation of the analogues from CD1d molecules and the affinity ofbinding to a soluble iNKT TCR.

We tested α-GalCer and(2S,3S,4R)-1-O-(α-_(D)-galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol(hereafter referred as to OCH), which has a shorter sphingosine chainthan α-GalCer and had previously been shown to bind to mouse CDmolecules with a shorter half-life, resulting in a weaker activation ofmouse iNKT cells (Miyamoto, Miyake et al, Nature, 2001, 413; 531-4, Oki,Chiba et al, 3 Clin Invest, 2004, 113; 1631-40).

In order to measure the rate of dissociation from CD1d molecules, wegenerated by phage display library a Fab antibody specific for CD1dmolecules loaded with α-GalCer (hereafter referred to as 9B Fab),Initial Biacore measurements and FACS staining of lipid pulsed C1R-CD1dcells demonstrated that the 9B Fab recognised specifically human CD1dmolecules loaded with all the tested compounds while it failed to stainunpulsed C1R-CD1d cells (data not shown). Using the 9B Fab we looked atthe rate of dissociation of all the tested compounds from soluble humanCD1d molecules using surface plasmon resonance. Biotinylated CD1d-lipidcomplexes were immobilized to streptavidin-coated chips and the level ofbinding of the 9B Fab was measured over time. The loss of binding of theantibody over time was used to determine the rate of lipid dissociationfrom CD1d molecules.

Results: OCH had a rate of dissociation 3.9 fold faster than α-GalCer,respectively (FIG. 1). These results were consistent with previouslypublished data, demonstrating that the stability of glycolipids bound toCD1d molecules depends on the length of the alkyl chains (Old, Chiba etal, J Clin Invest, 2004, 113; 1631-40).

We then assessed whether the length of the acyl and sphingosine chainscould affect the affinity of iNKT TCR binding to glycolipid-CD1dcomplex. We refolded Vα24 and Vβ11 chains of the iNKT TCR as previouslydescribed (Gadola, Koch et al, J Exp Med, 2006, 203; 699-710) and usedthe purified and refolded iNKT TCR in surface plasmon resonance studiesagainst immobilized biotinylated CD1d monomers loaded with eitherα-GalCer or OCH. The results of these experiments demonstrated that anincrease in the sphingosine chain length correlated with an increase inTCR binding affinity, suggesting that a reduction of the lipid chainlength negatively affects TCR binding affinity to lipid-CD Id complexes(FIG. 2).

Conclusions: The crystal structure of human CD1d-GalCer demonstratedthat α-GalCer exploits fully the binding capacity of CD1d. Using surfaceplasmon resonance, we found that: i) shortening of either alkyl chainssignificantly reduced the stability of CD1d/lipid complexes (FIG. 1 anddata not shown); ii) shortening of the sphingosine chain of α-GalCerreduces the iNKT cell TCR affinity by 1.00 fold (FIG. 2), resulting inchanges to the iNKT cell immunological synapse, polarization of iNKTcell cytotoxic granules and iNKT cell activation (data not shown). Incontrast, variations in either the length or saturation of the acylchain do not alter iNKT cell TCR affinity (data not shown). Analysis ofpreviously reported structures of empty and loaded human CD1d moleculessuggests that incomplete occupation of the binding groove by a shortenedsphingosine chain could result in conformational differences at the TCRrecognition surface. This indirect effect provides a general mechanismby which the length of the lipid chain occupying the CD1d C′ channelplays a role in controlling the affinity of lipid specific CD restrictedT cells.

The observed modulation of TCR binding affinity of the OCH/CD1d complexis therefore achieved at the cost of greater instability of the CD1dlipid complex, caused by the shortened sphingosine chain. Since NKT cellagonists with shorter sphingosine chains will have a shorter lifespan invivo, we aimed to define a new family of compounds capable of bindingwith high affinity to CD1d. Such new class of molecules would provide uswith an opportunity to fine-tune the range of binding affinity betweenthe NKT TCR and the CD1d/lipid complexes.

Modification of the Polar Head.

i. Glycerol Ceramide Derivatives

Lower Affinity of the Invariant TCR for α-GalCer Analogs.

To characterize the affinity of analogs of α-GalCer, biotinylated hCD1dmonomers were generated using previously described protocols(Karadimitris, Gadola et al, Proc Natl Acad Sci USA, 2001, 98; 3294-8)bound to the different analogs. Surface plasmon resonance (Biacore)analysis was performed using a soluble disulphide-linked human invariantVα24⁺/Vβ11⁺ TCR (Gadola, Koch et al, Exp Med, 2006, 203; 699-710) tomeasure the equilibrium dissociation constants (K_(d)) of binding of theTCR to different monomers (FIG. 3).

The K_(d) of the TCR binding to the α-GalCer containing monomer was1.29M, while that of threitolceramide was lower affinity at 5.74 μM.Binding of the TCR to the 4S and 4R threitolceramide monomers was ofslightly higher than to the unmodified threitolceramide at 3.84 μM and4.25 μM respectively.

Differences in the kinetic measurement of the rate of dissociation(K_(off)) between the TCR and the different monomers were of similarmagnitude between the analogs as in the equilibrium studies (FIG. 3)with 0.37 s⁻¹ for α-GalCer and 0.506 and 0.650 s⁻¹ for the 4R and 4Sthreitolceramide derivatives respectively. The quickest K_(off) of thosetested was with the unmodified threitolceramide analog at 1.04 s⁻¹indicative of a lower affinity interaction. It is tempting to speculatethat the difference in affinity observed between the 4S and 4R variantswas due to increased stability of the phenyl-threitol head groupinteracting with the TCR. These data suggest that the structurepreviously generated can be useful in the rational design ofCD1d-binding analogs that may have different properties to α-GalCer.

In an effort to identify the minimal residues required to stimulate iNKTcells, we decided to generate a family of compounds retaining highaffinity to CD1d molecules (i.e. with maximum length of both alkylchains), and containing either a 3-carbon (i.e. glycerol-mimic headgroup), a 4-carbon (i.e. threitol-mimic head group, hereafter referredas to threitol-ceramide) or a 5-carbon (i.e. arabinitol mimic headgroup).

Immature human DC were cultured in the presence of a human iNKT cellclone with or without analogs or vehicle at different concentrations.After 24 hours, DC were assessed by flow cytometry for the upregulationof different maturation markers. α-GalCer induced significantupregulation of all the markers examined (CD83, CD86 and CD38) (FIG. 4and unpublished data). In each case the degree of maturation observedwith an analog was reduced compared with α-GalCer. Threitolceramide wasmore potent than glycerolceramide and arabinitolceramide, whilemodifications in the phenyl-variants 4R-threitolceramide and4S-threitolceramide do not seem to have affected their function.Interestingly arabinitolceramide appeared to be only weakly functionalin this system, which may be significant and indicate the degree ofinteraction necessary to stimulate through the human invariant TCR.

Release of IL-12p40 (and bioactive p75) by DC is a marker of activationand is thought to play an important role in regulating the profile ofthe immune response generated in response to those DC (Trinchieri et al,Nat Rev Immunol, 2003, 3; 133-46). IL-12p40 was measured from thesupernatants of the mixed cultures and the concentration of 11-12p40released (FIG. 4B) reflected the maturation responses previouslydescribed (FIG. 4A). α-GalCer induced significant levels of IL-12p40,while threitolceramide and glycerolceramide induced approximately 50% ofthe level seen in the presence of α-GalCer. Both 4R and 4Sthreitolceramide induced a less potent response while little IL-12p40was detected in the presence of arabinitolceramide.

Similar responses were obtained when iNKT cell activation was examined.Release of IFN-γ by the iNKT cells in response to stimulation by analogspresented by DC induced approximately 40 ng/ml IFN-γ in the presence ofα-GalCer, 20 ng/ml with threitolceramide and glycerolceramide and nodetectable IFNγ when arabinitolceramide was added to the culture (FIG.4C).

From these assays, threitolceramide was found to be a potent, loweraffinity analog of α-GalCer. To compare the functions of high affinityα-GalCer, intermediate affinity threitolceramide with the known, lowaffinity analog OCH, the three compounds were titrated onto DC and usedto present to iNKT cells. Maturation of DC and DC viability wereexamined after 48 h. Although α-GalCer induced maximal DC maturation atonly 0.8 ng/ml compared with 67 ng/ml for threitolceramide or OCH, atthe same concentrations (FIG. 4D) only 10% of DC were still viable whenstimulated with α-GalCer, while a significantly greater proportion of DCwere propidium iodide negative when either analog was used (FIG. 4E).These data show that threitolceramide can induce good functionalresponses from iNKT cells in vitro, while maintaining significant DCviability compared with α-GalCer.

These data suggest that lower affinity analogs of α-GalCer can stimulatepotent iNKT-cell dependent DC maturation without the significant DCkilling observed with α-GalCer. Unlike OCH, threitolceramide should havethe same binding affinity for CD1d as the sphingosine and acyl chainsare identical to those in α-GalCer.

The following Experiments further exemplify potential uses of this newclass of CD1d ligands:

Stimulation of iNKT cells by α-GalCer in the context of CD1d rapidlyinduces release of significant levels of a number of cytokines includingIFN-γ and IL-4 (Burdin, Brossay et al, J Immunol, 1998, 161; 3271-81),IL-3 and GM-CSF (Leite-de-Moraes, Lisbonne et al, Eur J Immunol, 2002,32; 1897-904) with other downstream cell-types including DC (Kitamura,Iwakabe et al, J Exp Med, 1999, 189; 1121-8) and NK cells (Carnaud, Leeet al, J Immunol, 1999, 163; 4647-50) being activated. Analogs such asOCH (Miyamoto, Miyake et al, Nature, 2001, 413; 531-4) and 20:2 (Yu, Innet al, Proc Natl Acad Sci USA, 2005, 102; 3383-8) have been shown toinduce a modified cytokine response, with IL-4 release but little or noIFN-γ. Injection of glycerolceramide into wild type mice did not induceany detectable cytokine release (FIG. 5. B and C) as would be expectedby the lack of iNKT cell-dependent DC (FIG. 5A). However whenthreitolceramide or arabintolceramide were injected, IL-4 (FIG. 4B),IFN-γ (FIG. 5C) and IL-12p40/70 (data not shown) were detected in theserum with a similar time-course to that seen with α-GalCer. Bothcompounds were less potent than α-GalCer at equivalent doses. Neithercompound showed a skewed Th2 cytokine response as seen with OCH(Miyamoto, Miyake et al, Nature, 2001, 413; 531-4, Silk, Hermans et al,J Clin Invest, 2004, 114; 1800-11).

Adjuvant Function of α-GalCer Analogs Induces Effective Tumour SpecificT Cell Responses In Vivo.

Previously we, and others have shown that co-injection of model antigenssuch as OVA (Silk, Hermans et al, J Clin Invest, 2004, 114; 1800-11,Fujii, Shimizu et al, J Exp Med, 2003, 198; 267-79, Hermans, Silk et al,Immunol, 2003, 171; 5140-7) and β-galactosidase (Silk, Hermans et al, JClin Invest, 2004, 114; 1800-11) together with α-GalCer induces enhancedCD8⁺, CD4⁺ T cell and B cell responses. These were effective for therapyagainst tumour cells expressing the target antigen. The various analogswere co-injected with OVA into wild type mice and 6 days later the bloodexamined for the presence of antigen-specific CD8⁺ T cells usingfluorescent K^(b)-SIINFEKL tetramers. Both arabintolceramide andthreitolceramide induced a significant CD8⁺ T cell response after 6days, although less potently than α-GalCer, while glycerolceramide didnot (FIG. 6A). Sera from the mice were examined for the presence ofOVA-specific antibodies using ELISA and showed responses of similarranking order to those seen with CD8⁺ T cells (FIGS. 7B and 7C).

B Cell Responses.

Non-glycolipid analogs of α-GalCer stimulate B cell maturation in thepresence of iNKT cells in vivo and subsequent antibody production. (A)C57BL/6 or iNKT^(−/−) mice were injected i.v. with 1 μg of vehicle,α-GalCer, analog or 25 μg MPL. Twenty hours after injection splenocyteswere stained with antibodies against B220 and CD86 and analyzed by flowcytometry. Maturation was assessed by the upregulation of CD86 at thecell surface, gating on B cells (B220⁺) (FIG. 7 A). Mean fluorescenceintensity for each histogram is indicated. (B) Simultaneousadministration of 1 μg α-GalCer or analogs and 400 μg OVA inducessignificant OVA-specific IgGs. Mice were bled 11-14 d afteradministration and the serum tested by ELISA for antibodies (FIG. 7 B).

Priming of antigen specific T cell responses using human in vitropriming Model.

Experiments carried out with human peripheral blood lymphocytesdemonstrated that threitol-ceramide is superior than glycerol-ceramidein expanding Melan-A₂₆₋₃₅ specific T cell responses. (FIG. 8)

II. Inositol Derivative Analogs of α-GalCer

Subsequently, a new panel of novel α-GalCer analogs was generated. Thefirst was an inositolceramide with a 25-carbon fatty acid chain(INOC-25). INOC-25 induced detectable DC maturation and cytokineproduction from iNKT cells. (Data not shown). Further structuralmodifications were made to generate 6-Sulfono-myo-inositolceramide and6-Deoxy-myo-inositolceramide. These compounds were tested for theireffects on DC maturation in the human co-culture system (FIG. 9A) and inmice in vivo (unpublished data). While the inositol derivative INOC-25induced little DC maturation (data not shown) by upregulation of CD83and CD86, both 6-Deoxy and 6-Sulfono-myo-inositolceramide inducedsignificant DC maturation in an iNKT cell dependent manner (FIG. 9A.) toa similar extent to that observed with α-GalCer.

Similar to that observed with DC maturation (FIG. 9A.), IL-12p40production by the DC was almost identical between et-GalCer and6-Deoxy-myo-inositolceramide, while the 6-Sulfono derivative wasslightly weaker (FIG. 9B). However the amount of IL-12p40 produced bythe DC in response to iNKT cells stimulated by the 6-Deoxy and 6-Sulfonoderivative analogs was significantly above that from the INOC-25 (notshown) and also greater than that observed with LPS. The supernatantsfrom the DC co-cultures with the inositol derivative compounds wereexamined for IFN-γ as a measure of iNKT cell activation. While the6-Deoxy compound induced similar IFN-γ release to that observed withaddition of α-GalCer (FIG. 9C), 6-Sulfono-myo-inositolceramide inducedvery little IFN-γ release from the iNKT cells.

The three inositol compounds were titrated on C1R cells expressing humanCD1d and used to stimulate an iNKT cell clone. From both the IFN-γ (FIG.9D.) and the IL-4 release (FIG. 9E.) it appears that while the INOC-25induced little or no IFN-γ and IL-4, 6-Deoxy inositolceramide stimulatedrelease of significant levels of both cytokines from the iNKT cells.Interestingly, as observed when co-cultured with DC (FIG. 9C), while6-Sulfono inositolceramide stimulated little or no IFN-γ release (FIG.9D), detectable IL-4 was produced when higher concentrations of thecompound were used (FIG. 9E).

In comparison with α-GalCer, each of the inositol derivative compoundsappeared to have lower affinity. Both in terms of IFN-γ (FIG. 9C) andIL-4 (FIG. 9B) α-GalCer induces release of significantly higherconcentrations of cytokines. IFN-γ and IL-4 appeared to be approaching aplateau when stimulated with 200 ng/ml α-GalCer. Although IFN-γ and IL-4induced by the inositol derivatives also appear to be titrating withincreasing concentration of compound, they did not approach the levelsinduced by α-GalCer.

It is possible that while C1R cells are effective at presenting higheraffinity ligands such as α-GalCer, they are less effective at presentinglower affinity compounds than professional APCs such as DC. This mayexplain the difference in the magnitude of cytokine production observedbetween analogs presented by DC and by CD1d-transfected C1R cells.

Conclusions: Fine tuning the affinity of binding of the iNKT TCR to theCD1d/lipid complex and the stability of lipid ligands to CD1d moleculeshas led to the identification of a new group of compounds capable ofinducing iNKT cell activation, without over-stimulating iNKT cells. Thisnovel class of compounds is efficient in stabilizing CD1d molecules andit is optimized for NKT stimulation since it minimizes NKT celldependent DC lysis and cytokine release, while ensuring DC maturationand antigen-specific T and B cell priming. This novel combination ofuseful molecular and biological attributes was achieved by designing aclass of CD1d ligands that optimize the iNKT/CD1d interaction withoutcompromising the stability of the CD1d/ligand complex. Such compoundscan therefore be used in broader effective dose range than existingcompounds.

EXAMPLE A

These experiments describe the priming of dendritic cells (“DC”shereafter). In this, and the following examples, Compound 1, arabinitolceramide; Compound 2, glycerol ceramide; or Compound 3, threitolceramide derivatives) were used.

The methodology of Salio, et al., J. Immunol., 167:1188-1197 (2001),incorporated by reference, was followed. In brief, 2×105 human monocytederived DCs were pulsed with 100 ng/ml of one of Compounds 2, 3,αGal-Cer, or vehicle solution, as a control. For DC pulsing, each ofarabinitol ceramide (Compound 1), threitol ceramide (Compound 3),glycerol ceramide (Compound 2) and αGal-Cer was solubilized in vehiclesolution (0.5% Tween 20/PBS), and added to the medium of the DCcultures. NKT cells were also added to the DC cultures at a ratio of 10DC/NKT cell. After 36 hours, supernatant was removed from cultures andanalyzed for the presence of known DC maturation markers CD83, CD80,CD86, CD25, and CD38, all of which were analyzed via FACS, using wellknown methods.

The results indicated that when DCs are combined with NKTs in thepresence of either Compound 1 (arabinitol ceramide) or Compound 2(glycerol ceramide) or Compound 3 (threitol ceramide) DC maturationmarkers are induced. Levels of maturation marker induction are similarto those seen with αGal-Cer. No upregulation was seen with controls.

In addition, interleukin-12 levels (IL-12, were measured as anindication of DC stimulation by detecting the p40 form of IL-12 viaELISA, using standard methods. As NKT cells do not produce IL-12 thisindicates a DC specific response. All compounds produced secretion ofIL-12 in this assay. Compound 3 was seen to be approximately 50% aspotent as αGal-Cer in this assay whilst Compounds 1 and 2 wereapproximately 1000 fold less potent than Compound 3 confirming differingbiological activities of these compounds in this assay system.

EXAMPLE B

In these experiments, T cell expansion was measured, by pulsing humanDCs from PBLs with 100 nM of the known, immunogenic peptide MelanA26-35,ELAGIGILTV. The peptide was added to the DCs together with syngeneicPBLs from the same donor. To detail these experiments more fully,samples of DCs were irradiated with 3000 rads followed by a 3 hour pulsewith the peptide, in serum free medium.

The cells were then washed thoroughly and incubated with autologous,PBLs, at a 1:10 ratio, in RPMI 1640/5% human serum. Recombinant humanIL-2 was added at 10 U/ml from day 4 to day 7. At day 10, following theaddition of the PBLs, cells were analyzed. Cultured cells were stainedwith anti-CD8 and A2/MelanA2S-35 tetramer. The percentage of MelanAspecific CD8 T cells out of total CD8 positive cells was measured byFACS analysis.

The results indicated that when human DCs were incubated in the presenceof NKT cells, threitol and glycerol cross-priming occurs and that bothcompounds were more potent stimulators of antigen specific CTLresponses, in vitro, than αGal-Cer. This confirms that both Compound 2,glycerol ceramide and Compound 3, threitol ceramide are likely to beuseful as CD8+ CTL immunostimulants and suggests that other compounds ofthe invention may behave similarly.

EXAMPLE C

These animal experiments detail work designed to study the impact of thecompounds of the invention in vivo.

Subject animals, five mice per group, received an intravenous injectionof 400 μg of ovalbumin (OVA) together with 1 μg of one of the compoundsof the invention under examination or an equivalent volume of PBSdiluted vehicle solution. A subset of mice were injected with 25 μg ofthe molecule MPL (Sigma-Aldrich; extracted from Salmonella Minnesota)solubilized in PBS.

Following the injections, blood was obtained from lateral tail veins,and PBLs were isolated, using standard methods. The PBLs were thenstained, directly, in vivo, with tetrameric, H-2 Kb/OVA 257-264, H-2 Kbcomplexes, following Palmowski, et al., J. Immunol., 168:4391-4398(2002), incorporated by reference. The tetramers were prepared inaccordance with Whilan, et al., J. Immunol., 163:4342-4348 (1999),incorporated by reference.

When the PBLs from naïve animals were compared to those from animalsstained with irrelevant tetramers the background stainings wereequivalent.

There was an enhancement of the OVA specific response when αGal-Cer,Compound 1, arabinitol ceramide, or Compound 3, threitol ceramide wereused. Enhancement was weakest with Compound 2, glycerol ceramide.

When MPL was coinfected with either αGal-Cer or Compound 3, threitolceramide, there was an enhancement of the CTL response as might beexpected from Silk et al., J. Clin. Invest., 114:1800-1811, 2004.

EXAMPLE D

These experiments assessed the presence of OVA specific IgG antibodiesin the serum of mice which had been injected with the OVA protein.

OVA protein coated ELISA plates, were prepared, using well knownmethods. Then, blood samples were taken, ten days followingimmunization, and serum was prepared.

The samples were taken from mice that had been immunized with OVA only,OVA and Compound 1, arabinitol ceramide, OVA and αGal-Cer, or OVA andCompound 2, glycerol ceramide. Additionally, mice were immunized witheach of these schedules with MPL added. Serum IgG titers were measuredby adding serial dilutions of mouse serum were detected usinghorseradish peroxidase (HRP) conjugated α-mouse IgG.

The data indicated that the compounds of the invention produce OVAspecific antibody responses. Compound 1, arabinitol ceramide andαGal-Cer were seen to be more effective as adjuvants than MPL whereasCompound 2, glycerol ceramide, was less effective than MPL whilst stillproducing OVA specific antibodies.

Additionally, when MPL was added strong enhancement of the response wasseen in all cases demonstrating that compounds of the invention whenused in combination with a TLR4 ligand are extremely efficient inexpanding the titer of antigen specific antibodies.

EXAMPLE E

In these experiments, the DCs obtained from the spleen of subjectanimals were phenotyped.

In brief, in each experiment, 1 μg of one of a selection of compounds ofthe invention was injected into the tail vein of subject mice. 24 hlater spleen tissue was removed from the animals and gently teasedthrough gauze into complete medium, supplemented with 5 mM of EDTA.Cells were enriched for CD11c by anti-CD11, magnetic beads usingpublished techniques. Once enriched, they were assayed for maturationmarkers using antibody staining and flow cytometry. Non-specificstaining was addressed by using an antibody specific for the Fc-RIII/IIreceptor.

Compound 1, arabinitol ceramide, and Compound 2, threitol ceramide andαGal-Cer all induced CD86 in vivo similarly, confirming that both thesecompounds in mice are highly efficient at producing T cell as well as Bcell responses. In contrast, Compound 3, glycerol ceramide, did notinduce the DC86 marker significantly. These results show that while theMPL and glycerol combination is effective in driving antigen specific Bcell responses (Examples 25 and 26) in mice, DC maturation and T cellexpansion do not appear to be significantly enhanced by Compound 2,glycerol ceramide. This contrasts to the finding in humans whereCompound 2, glycerol ceramide, matures DC cells in the presence of NKTcells which then serve as efficient primers of human CTLs.

EXAMPLE F

These experiments were performed to determine the effect of selectedcompounds of the invention in stimulating the production of IL-12production in subject animals. The animals received 1 μg or 10 μg ofthreitol, glycerol or Gal-Cer. One group of animals also received 50 μgof MPL. Six hours following injection, tail blood samples were collectedand the IL-12 p70 protein was determined using an ELISA all inaccordance with standard procedures.

Dose dependent increases in IL-12/p70 secretion were observed withCompound 3, threitol ceramide treated mice, confirming the potency ofthis molecule in inducing IL-12/p70 release. The effectiveness ofCompound 3 compares extremely favorably with MPL.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Compound 1: Arabinitol Ceramide

(a) L(+) arabinose was converted into the arabinitol triflate compound,i.e.

in accordance with well known procedures. See, Zinner, et al., Chem.Ber., 92:1614 (1959); Qin, et al., Can. J. Chem., 77:481 (1999); and,Yann, et al., Carbohydr. Res., 74:323 (1979), all incorporated byreference. This compound was combined with a sphingosine component,i.e.,

which was synthesized from 4,6-O-benzylidene-D-galactose (Gros, et al.,J. Org. Chem., 29:3647 (1941)), following any of Zimmermann, et al.,Liebigs Ann. Chem., 663 (1988); Schmidt, et al., Carbohydr. Res.,172:169 (1988) and Figueroa-Perez, et al., Carbohydr. Res., 328:95(2000).

Once these two compounds were available, a solution of the sphingosinecomponent (220 mg, 0.575 mmol), in 3 ml of anhydrous THF, and 95% NaH(17 mg, 0.708 mmol) was added at 0° C. After 15 minutes of stirring, asolution of the arabinitol triflate compound (251 mg, 0.690 mmol), in 2mL, of anhydrous THF was added at the same temperature. The resultingmixture was warmed, slowly, to room temperature, and then stirredovernight. The reaction mixture was quenched with an aqueous solution ofNH₄Cl, taken up into EtOAc, and the layers were separated.

The organic layer was washed with water, dried over anhydrous MgSO₄, andevaporated to dryness. Crude material was purified via flashchromatography (1:9, ethylacetate:petroleum ether, yielding:

as a colourless liquid (yield: 95%). R_(f)=0.54 (1:9, ethylacetate:petroleum ether). [α]_(D) ²⁵=+5.2 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 4.19-4.00 (m, 5H), 3.99-3.86 (m, 2H), 3.84-3.61 (m, 5H),1.60-1.25 (m, 26H), 1.41 (s, 3H), 1.40 (br s, 6H), 1.38 (s, 3H), 1.33(s, 3H), 1.30 (s, 3H), 0.87 (t, J=6.5 Hz, 3H). ¹³C NMR (62.5 MHz,CDCl₃): δ 109.6, 108.2, 96.0, 79.7, 77.7, 77.07, 77.3, 75.7, 72.8, 71.8,67.5, 60.0, 31.9, 29.6, 29.5, 29.66, 29.62 29.59, 29.56, 29.4, 29.3,28.1, 26.9, 26.6, 26.4, 25.6, 25.2, 22.6, 14.1. MALDI-MS (positive mode,Matrix CHCA): m/z 620.2 [M+Na]⁺. Anal. Calcd for C₃₂H₅₉N₃O₇ (597.43): C,64.29; H, 9.95; N, 7.03. Found: C, 64.35; H, 10.01; N, 7.15

(b) The product of (a) above (150 mg, 0.251 mmol), and 10% Pd/C (100 mg)in 4 ml methanol with a drop of acetic acid was stirred, under an H₂atmosphere, for 20 hours, at room temperature. Then the mixture wasfiltered, concentrated, and co-evaporated with toluene. The resultingsyrup was dissolved in 5 ml of dry DMF, and then hexacosanoic acid(Fluka) (120 mg, 0.303 mmol), N-hydroxy-benzotriazole (40 mg, 0.301mmol), and 1-[3-(dimethylamino-propyl]-3 ethylcarbodiimide hydrochloride(EDC, 58 mg, 0.303 mmol) were added successively, and the resultingmixture was stirred at 45° C. for 1 day. The mixture was taken in ethylacetate washed with water, saturated brine solution, dried overanhydrous MgSO₄, and concentrated. Residue was purified by flashchromatography to yield 182 mg of

as a colourless solid (yield: 75%). mp 89° C. R_(f)=0.46 (2:8, ethylacetate:petroleum ether). [α]_(D) ²⁵=+11.4 (c 1.0, CHCl₃). NMR (250 MHz,CDCl₃): δ 5.84 (d, J=8.5 Hz, 1H), 4.25-4.00 (m, 1H), 3.98-3.92 (m, 1H),3.85-3.74 (m, 2H), 3.70-3.62 (m, 1H), 3.56-3.49 (m, 2H), 2.17-2.11 (m,2H), 1.60-1.24 (m, 72H), 1.41 (s, 3H), 1.38 (s, 6H), 1.36 (s, 3H), 1.32(br s, 6H), 0.86 (t, J=6.7 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.4,109.6, 107.8, 79.8, 77.9, 77.8, 77.1, 76.1, 72.4, 71.3, 67.6, 48.3,36.9, 29.69, 29.64, 29.5, 29.3, 29.0, 28.0, 27.1, 27.0, 26.7, 26.4,25.77, 25.72, 25.2, 22.6, 14.0. MALDI-MS (positive mode, Matrix CHCA):m/z 972.6 [M+Na]⁺. Anal. Calcd for C₅₈H₁₁₁NO₈ (949.83): C, 73.29; H,11.77; N, 1.47. Found: C, 73.59; H, 11.99; N, 1.41

(c) The product of (b) above (120 mg, 0.123 mmol) in MeOH:CH₂Cl₂ (10:1,22 ml) containing TFA (100 μl), was stirred at room temperature for 3days. The solid resulting was filtered and dried to give

hereinafter referred to as Compound 1 (‘arabinitol-ceramide’), 63 mg,60% yield, mp 125° C., ¹H NMR (400 MHz, C₅D₅N): δ 8.66 (d, J=8.8 Hz,1H), 5.30-5.25 (m, 2H), 4.98-4.94 (m, 1H), 4.66-4.58 (m, 2H), 4.44-4.22(m, 2.57-2.53 (m, 2H), 2.07-1.37 (m, 72H), 0.99 (t, =6.7 Hz, 6H). ¹³CNMR (150.9 MHz, C₅D₅N): δ 173.3, 78.1, 76.3, 74.6, 73.2, 72.7, 71.3,70.0, 65.4, 51.8, 36.8, 32.1-29.6 (in), 26.4, 22.9, 14.1. MALDI-MS(positive mode, Matrix CHCA): m/z 853.3 [M+Na]⁺. Anal. Calcd forC₄₉H₉₉NO₈ (830.73): C, 70.88; H, 12.02; N, 1.69. Found: C, 73.59, H,11.99; N, 1.41.Compound 2: Glycerol Ceramide

The procedure of 1(a) above was repeated using

as the triflate starting material (Cassel, et al., Eur. J. Org Chem.,875 (2001)) to give

as a colourless solid (yield: 90%). R_(f)=0.47 (1:9, ethylacetate:petroleum ether). [α]_(D) ²⁵=+24.6 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 4.32-4.23 (m, 1H), 4.16-4.04 (m, 2H), 3.96 (d, J=7.8 Hz,1H), 3.87-3.76 (m, 2H), 3.68-3.48 (m, 4H), 1.57-1.18 (m, 26H), 1.42 (s,3H), 1.40 (s, 3H), 1.36 (s, 3H), 1.30 (s, 3H), 0.87 (t, J=6.5 Hz, 3H).¹³C NMR (62.5 MHz, CDCl₃): δ 109.3, 108.2, 77.7, 75.6, 74.5, 72.8, 72.4,66.7, 59.8, 31.8, 29.6, 29.5, 29.4, 29.3, 29.1, 28.0, 26.7, 26.3, 25.6,25.3, 22.6, 14. 1. MALDI-MS (positive mode, Matrix CHCA): m/z 520.1[M+Na]⁺. Anal. Calcd for C₂₇H₅₁N₃O₅ (497.38): C, 65.16; H, 10.33; N,8.44. Found: C, 65.25; H, 10.41; N, 8.50.

(b) The procedure of 1(b) was repeated using product of (a) above togive

as a colorless solid (yield 72%). mp 81° C. R_(f)=0.43 (2:8, ethylacetate:petroleum ether). [α]_(D) ²⁵=+13.5 (c 1.0, CHCl₃). ¹H NMR (400MHz, CDCl₃): δ 5.75 (d, J=9.0 Hz, 1H), 4.27-4.00 (m, 5H), 3.74 (dd,J=9.3, 3.1 Hz, 1H), 3.69 (dd, J=8.2, 6.2 Hz, 1H), 3.54-3.45 (m, 3H),2.20-2.07 (m, 2H), 1.60-1.23 (m, 72H), 1.40 (s, 6H), 1.34 (s, 3H), 1.31(s, 3H), 0.86 (t, J=6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 172.4,109.4, 107.8, 77.7, 75.9, 74.7, 72.5, 66.4, 48.1, 36.9, 31.8, 29.6,29.5, 29.39, 29.34, 29.2, 28.9, 27.9, 26.7, 26.4, 25.7, 25.3, 22.6,14.1. MALDI-MS (positive mode, Matrix CHCA): m/z 873.2 [M+Na]⁺. Anal.Calcd for C₅₃H₁₀₃NO₆ (849.77): C, 74.86; H, 12.21; N, 1.65. Found: C,74.81; H, 12.26; N, 1.70.

(c) The procedure of 1(c) above was repeated using the product of (b)above to give

as a colorless solid, hereinafter referred to as Compound 2 (‘glycerolceramide’). Yield 70%, mp 140° C. ¹H NMR (400 MHz, C₅D₅N): δ 8.64 (d,J=8.67 Hz, 1H), 4.92-4.83 (m, 1H), 4.60-4.56 (m, 1H), 4.41-4.31 (m, 2H),4.12-3.96 (m, 6H), 2.57 (t, J=7.2 Hz, 2H), 2.03-1.37 (m, 72H), 0.99 (t,J=6.8 Hz, 6H). MALDI-MS (positive mode, Matrix CHCA): m/z 793.6 [M+Na]⁺.Anal. Calcd for C₄₇H₉₅NO₆ (769.72): C, 73.29; H, 12.43; N, 1.82. Found:C, 73.33; H, 12.47; N, 1.91.Compound 3: Threitol Ceramide

Compound (100 mg, 0.4 mmol) as prepared according to in accordance withwell known procedures (see, Wagner, et al., J. Chem. Soc., Perkin Trans.1, 780 (2001))

was added to 2,6-di-tert-butylpyridine (92 mg, 0.48 mmol) in anhydrousCH₂Cl₂ mL), Tf₂O (0.08 mL, 0.48 mmol) dissolved in anhydrous CH₂Cl₂ (1mL) with stirring at 0° C. The reaction mixture was stirred at the sametemperature for 1 hour. The reaction mixture was taken in ethyl acetateand washed with cold water (2×15 mL). The combined organic layers werewashed with brine solution, dried and evaporated to get the crudeproduct which was purified by silica gel column chromatography (1:10,ethyl acetate:petroleum ether containing drops of Et₃N) to give

(145 mg, 95%). R_(f) 0.41 (1:10, ethyl acetate:petroleum ether). NMR(250 MHz, CDCl₃): δ 7.41-7.27 (m, 5H), 4.71 (dd, J=10.9, 2.8 Hz, 1H),4.57 (s, 2H), 4.50 (dd, J=10.9, 4.9 Hz, 1H), 4.14 (ddd, J=8.0, 4.9, 2.8Hz, 1H), 4.05 (ddd, J=8.1, 6.3, 4.5 Hz, 1H), 3.73 (dd, J=9.7, 4.5 Hz,1H), 3.55 (dd, J=9.7, 6.3 Hz, 1H), 1.42 (s, 6H). ¹³C NMR (62.5 MHz,CDCl₃): δ 128.6, 128.5, 128.0, 127.7, 110.7, 76.8, 75.03, 75.00, 73.8,69.9, 26.9, 26.6.

(b) The procedure of 1(a) above was followed using the product of (a)above to give

which was purified by flash chromatography (1:9, ethyl acetate:petroleumether) yielding a colorless liquid (306 mg, 94%). R_(f)=0.46 (1:9, ethylacetate:petroleum ether). [α]_(D) ²⁵=+7.3 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.36-7.23 (m, 5H), 4.50 (s, 2H), 4.17-3.98 (m, 3H), 3.94(dd, J=9.8, 2.0 Hz, 1H), 3.83 (dd, J=9.2, 5.6 Hz, 1H), 3.70-3.53 (m,6H), 1.62-1.21 (m, 26H), 1.43 (s, 6H), 1.40 (s, 3H), 1.30 (s, 3H), 0.88(t, J=6.2 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 138.0, 128.3, 127.63,128.60, 109.6, 108.2, 77.8, 77.5, 77.4, 75.7, 73.5, 72.9, 72.2, 70.6,60.0, 31.9, 29.7, 29.62, 29.57, 20.5, 29.4, 29.3, 28.1, 27.0, 26.4,25.6, 22.7, 14.1, 4.8. MALDI-MS (positive mode, Matrix DHB): m/z 640.9[M+Na]⁺.

(c) The procedure of 1(b) above was followed using the product of (b)above to yield

which was purified by flash chromatography (4:6, ethyl acetate:petroleumether) to yield a colorless solid (116 mg, 81%). R_(f)=0.23 (3:7, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 5.74 (d, I=8.7 Hz,1H), 4.27-4.14 (m, 1H), 4.13-3.99 (m, 3H), 3.93-3.87 (m, 1H), 3.81 (3.55(m, 6H), 2.20-2.13 (m, 2H), 1.65-1.20 (m, 72H), 1.42 (s, 9H), 1.33 (s,3H), 0.88 (t, J=6.3 Hz, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.6, 109.2,107.9, 79.2, 77.7, 76.7, 76.2, 71.9, 71.6, 62.4, 48.1, 36.9, 31.9, 29.6,29.5, 29.4, 29.3, 29.0, 27.9, 26.9, 26.4, 25.7, 25.6, 22.6, 14.4, 14.0.MALDI-MS (positive mode, Matrix DHB): m/z 902.4 [M+Na]⁺.

(d) The procedure of 1(c) above was repeated using the products of (c)above to yield

a colorless solid (71 mg, yield 71%), hereinafter referred to asCompound 3 (‘threitol ceramide’). ¹H NMR (250 MHz, C₅D₅N): δ 8.62 (d,J=8.6 Hz, 1H), 4.81-4.05 (m, 9H), 2.52-2.39 (m, 2H), 2.35-1.15 (m, 72H),0.87-0.83 (m, 6H). MALDI-MS (positive mode, Matrix CHCA): m/z 822.9 [MNa]⁺.Compound 4: Threitol Ceramide C₁₅ acyl

(a) The product of 3(b) above e.g.

(175 mg, 0.284 mmol) and 10% Pd/C (1.50 mg) in methanol (3 mL)containing a drop of acetic acid was stirred under H₂ atmosphere(balloon) at room temperature for 22 hours. Then the mixture wasfiltered, concentrated and co-evaporated with toluene. The resultingsyrup was dissolved in dry DMF (4 mL). Palmitic acid (Fluka) (73 mg,0.284 mmol), N-hydroxybenzotriazole (38 mg, 0.284 mmol) and1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (54 mg,0.284 mmol) were added successively and the resulting mixture wasstirred at 45° C. for 1 clay. Then it was taken in ethyl acetate washedwith water, saturated brine solution, dried over anhydrous MgSO₄, andconcentrated. The residue was purified by silica gel chromatography(4:6, ethyl acetate:petroleum ether) to yield

(168 mg, 80%). R_(f)=0.16 (7:3 ethyl acetate:petroleum ether). [α]_(D)²⁵=+11.6 (c 1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 5.77 (d, J=9.4 Hz,1H), 4.27-4.14 (m, 1H), 4.13-3.99 (m, 3H), 3.93-3.87 (m, 1H), 3.81-3.55(m, 6H), 2.20-2.13 (m, 2H), 1.75-1.20 (m, 52H), 1.42 (s, 9H), 1.33 (s,3H), 0.88 (t, J=6.4 Hz, 6H). MALDI-MS (positive mode, CHCA): m/z 762.9[M+Na]⁺. 778.8 [M+K]⁺.

(b) The product of (a) above (120 mg, 0.162 mmol) is MeOH/CH₂Cl₂ (10:1,22 mL) containing TFA (100 μL) was stirred at room temperature for 65hours. The solid that dropped out, was filtered and dried to obtain

(70 mg, yield 65%) hereinafter referred to as Compound 4, (‘threitolceramide C15 acyl’) ¹H NMR (250 MHz, DMSO-d₆): δ 5.45-5.35 (m, 1H),4.60-3.75 (m, 10H), 2.04-1.89 (m, 2H), 1.57-1.02 (m, 52H), 0.83-0.72 (m,6H). MALDI-MS (positive mode, CHCA): m/z 682.8 [M+Na]⁺, 698.6 [M+K]⁺.

Synthesis 22-(Z)-Hexacosanoic acid

(a) THP-protected-11-bromoundecanol (10.0 g, 29.85 mmol) e.g.

and magnesium (1.0 g, 41.66 mmol) in dry THF (150 mL), were heated underreflux for a 3-4 hour period to make the Grignard reagent. This wasadded to 11-bromoundecanoic acid (8.0 g, 30.22 mmol) e.g.

in dry THF (100 mL) under argon at −20° C. Methylmagnesium chloride inTHF (−10.2 mL, 3 M) was added until cessation of gas evolution stopped,Li₂CuCl₄ was then added, stirring at −20° C. continued for 1 hour, andthen temperature was allowed to rise to room temperature. After 15hours, the reaction mixture was carefully neutralized with 10% H₂SO₄ andextracted with ethyl acetate (2×150 mL). The organic layer was driedover MgSO₄, and concentrated to give colourless solid, which waspurified by flash chromatography (1:9 ethyl acetate:petroleum ether)give

(10.9 g, 82% yield). This compound was dissolved in THF (50 mL) andesterified with diazomethane. The solvent was evaporated, and purifiedby chromatography (5:95 ethyl acetate:petroleum ether) to give the esterof THP-protected-11-bromoundecanol e.g.

as a colourless solid (10.9 g, 97% yield). R_(f)=0.65 (5:95, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 4.56 (t, J=3.3 Hz,1H), 3.90-3.81 (m, 1H), 3.76-3.65 (m, 1H), 3.65 (s, 3H), 3.52-3.45 (m,1H), 3.40-3.31 (m, 1H), 2.28 (t, J=7.5 Hz, 2H), 1.62-1.51 (m, 10H), 1.23(br. s, 321-1). ¹³C NMR (62.5 MHz, CDCl₃): δ 174.3, 98.8, 67.7, 62.3,51.4, 34.1, 30.7, 29.7, 29.68, 29.63, 29.60, 29.49, 29.44, 29.2, 29.1,26.2, 25.5, 24.9, 19.6. MALDI-MS (positive mode, CHCA): m/z 477.3[M+Na]⁺.

(b) The product of (a) above (8.0 g, 17.62 mmol) in methanol (200 mL)was treated with p-toluenesulfonic acid (6.7 g, 35.26 mmol). Thereaction mixture was stirred for 3-4 hours. After completion ofreaction, (TLC monitoring) solvent was evaporated and the residue wasdissolved in chloroform and washed with saturated NaHCO₃ solution. Thechloroform layer was dried and concentrated to give colourless solid,which was recrystallized (petroleum ether/ethyl acetate) to give

as colourless crystals (6.4 g, 98% yield). R_(f)=0.21 (2:8, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 3.66 (s, 3H), 3.63(t, J=6.5 Hz, 2H), 2.30 (t, J=7.5 Hz, 1H), 1.64-1.53 (m, 4H), 1.25 (br.s, 32H). ¹³C NMR (62.5 MHz, CDCl₃): δ 174.3, 63.0, 51.4, 34.1, 32.8,29.6, 29.59, 29.58, 29.4, 29.2, 29.1, 25.7, 24.9. MALDI-MS (positivemode, CHCA): m/z 393.8 [M+Na]⁺.

(c) The product of (b) above (6.0 g, 16.21 mmol) in dry CH₂Cl₂ (200 mL),was treated with Dess-Martin periodinane (DMP) (13.74 g, 32.42 mmol).The reaction mixture was stirred for 3-4 hours at room temperature.After completion of reaction, solvent was evaporated; residue wassuspended in diethyl ether and filtered off. The filtrate was washedwith NaHCO₃ solution, then brine solution. The ether layer was dried andconcentrated to give solid, which was purified by flash chromatography(5:95, ethyl acetate:petroleum ether) to give

as a colourless solid (5.5 g, 92% yield). R_(f)=0.62 (5:95, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 9.73 (t, J=1.8 Hz,1H), 3.63 (s, 3H), 2.39 (dt, J=14.0, 7.5, 2.0 Hz, 2H), 2.26 (t, J=7.5Hz, 2H), 1.66-1.56 (m, 4H), 1.21 (br. s, 32H). ¹³C NMR (62.5 MHz,CDCl₃): δ 202.8, 174.2, 51.3, 43.8, 34.0, 29.65, 29.61, 29.5, 29.49,29.42, 29.40, 29.3, 29.2, 29.1, 24.9, 22.0. MALDI-MS (positive mode,CHCA): m/z 391.9 [M+Na]⁺.

(d) A suspension of n-butyltriphenylphosphonium bromide (8.66 g, 21.73mmol) in dry THF (80 mL) under argon at −78° C., was treated with sodiumbis(trimethylsilyl) amide (21.73 mL, 1.0 M). The reaction mixture wasstirred for 15 minutes, at the same temperature, before a solution ofthe product from of (c) above (5 g, 13.58 mmol) in dry THF (30 mL) wasadded, and then temperature was allowed to rise to room temperature.After completion of reaction, it was quenched with aqueous NH₄Clsolution and then extracted with diethyl ether (2×80 mL). The etherlayer washed with brine, dried and evaporated to give residue, which waspurified by flash chromatography (5:95, ethyl acetate:petroleum ether)to give

as a colourless solid (5.42 g, 98% yield). R_(f)=0.65 (5:95, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 5.38-5.33 (m, 2H),3.66 (s, 3H), 2.30 (t, 7.5 Hz, 2H), 2.04-196 (m. 4H), 1.67-1.56 (m, 2H),1.40-1.25 (m, 36H), 0.90 (t, J=7.5 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ174.3, 130.0, 129.5, 51.3, 34.1, 29.7, 29.69, 29.64, 29.59, 29.56, 29.4,29.3, 29.28, 29.25, 29.1, 27.2, 24.9, 22.8, 13.7. MALDI-MS (positivemode, CHCA): m/z 431.8 [M+Na]⁺.

(e) The product of (d) above (2 g, 4.893 mmol) and sodium hydroxide (2.3g, 5.872 mmol) in methanol was heated to reflux for 2 hours aftercooling with ice, the precipitate was collected, suspended in water andacidified with concentrated hydrochloric acid (˜pH 1). The product wasfiltered off and recrystallized from glacial acetic acid to give22-(Z)-hexacosanoic acid e.g.

as colourless crystals (1.9 g, 98%). ¹H NMR (250 MHz, CDCl₃): δ 12.11(s, 1H), 5.38-3.33 (m, 2H), 2.26 (t, J=7.5 Hz, 2H), 2.02-1.99 (m, 4H),1.64-1.58 (m, 2H), 1.38-1.26 (m, 36H), 0.90 (t, J=7.5 Hz, 3H). ¹³C NMR(62.5 MHz, CDCl₃): δ 176.6, 129.8, 129.3, 33.9, 29.5, 29.49, 29.41, 29.38, 29.35, 29.31, 29.2, 29.1, 29.0, 28.9, 26.9, 24.7, 22.6, 13.5.Compound 5: Threitol-22-(Z)-Ceramide

(a) The procedure of 1(b) above was repeated using 22-(Z)-hexacosanoicacid and the product of 3(b) e.g.

to give

as a colourless solid (yield: 68%). R_(f)=0.23 (3:7, ethylacetate:petroleum ether). [α]_(D) ²⁵=12.7 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 5.71 (d, J=9.5 Hz, 1H), 5.35-5.31 (m, 2H), 4.21-3.97 (m,4H), 3.91-3.84 (m, 1H), 3.78-3.63 (m, 5H), 3.59-3.52 (m, 1H), 2.35 (br.t, J=6.7 Hz, 1H), 2.14 (dt, J=10.5, 7.5, 3.0 Hz, 2H), 2.01-1.94 (m, 4H),1.61-1.22 (m, 76H), 0.90-0.82 (m, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ172.5, 130.0, 129.5, 109.2, 107.9, 79.1, 77.7, 76.6, 76.1, 71.8, 71.6,62.3, 48.1, 36.8, 31.8, 29.66, 29.60, 29.5, 29.37, 29.32, 29.28, 29.26,29.23, 28.9, 27.8, 27.1, 26.9, 26.4, 25.69, 25.64, 22.8, 22.6, 14.0,13.7. MALDI-MS (positive mode, DHB): m/z 902.3 [M+Na]⁺. Anal. Calcd forC₅₄H₁₀₃NO₇ (877.77): C, 73.84; H, 11.82; N, 1.59. Found: C, 73.97; H,11.96; N, 1.67.

(b) The procedure of 1(c) above was followed using the product of (a)above to give

as a colourless solid, hereafter referred to as Compound 5(‘Threitol-22-(Z)-Ceramide’) (yield: 81%). ¹H NMR (250 MHz, C₅D₅N): δ8.48 (d, J=8.7 Hz, 1H), 5.39-5.30 (m, 2H), 4.39-4.35 (m, 1H), 4.42-4.08(m, 8H), 3.97-3.95 (m, 2H), 2.32 (t, J=7.5 Hz, 2H), 1.99-1.87 (m, 4H),1.81-1.64 (m, 4H), 1.77-1.12 (m, 60H), 0.78-0.71 (m, 6H). MALDI-MS(positive mode, Matrix CHCA): m/z 820.8 [M+Na]. Anal. Calcd forC₄₈H₉₅NO₇ (797.71): C, 72.22; H, 12.00; N, 1.75: C, 72.29; H, 11.93; N,1.80.Compound 6: 4-Deoxy-4-Phenyl-Threitol-Ceramide

(a) Compound

was prepared with well known procedures (see, Su, et al., Tetrahedron,57:2147 (2001)); Surivet, et al., Tetrahedron Lett., 39:7299 (1998) andSurivet, et al., Tetrahedron, 55:1311 (1999)). The above compound (1 g,3.048 mmol) in CH₂Cl₂ (20 mL) was treated with pyridine (1.48 mL, 18.291mmol) and 4-dimethylamino pyridine (4-DMAP) (110 mg, 0.901 mmol)followed by phenoxythiocarbonyl chloride (0.630 mL, 4.56 mmol) at roomtemperature. After 30 min, the reaction mixture was diluted with CH₂Cl₂,washed with 10% NaHCO₃ solution, water, dried over MgSO₄ andco-evaporated with toluene to give residue. The crude product wasdissolved in toluene (30 mL), tributyltin hydride (2.45 mL, 9.144 mmol)and AIBN (150 mg, 0.914 mmol) were added, and the reaction mixture wasrefluxed under argon atmosphere for 4 hours. Concentrated and purifiedby flash chromatography (1:9, ethyl acetate:petroleum ether) to give thereduction product

as a colourless liquid (900 mg, 95% yield). R_(f)=0.40 (1:9, ethylacetate:petroleum ether). [α]_(D) ²⁵=−10.2 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.31-7.13 (m, 10H), 3.99 (br. s, 2H), 4.01-3.93 (m, 2H),3.87-3.80 (m, 2H), 3.31-3.18 (m, 2H), 2.92 (dd, 13.7, 6.5 Hz, 1H), 2.77(dd, J=13.7, 6.5 Hz, 1H), 1.31 (s, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ137.9, 137.2, 129.3, 128.2, 127.5, 126.4, 108.9, 79.6, 78.4, 76.4, 73.3,70.3, 39.4, 27.1, 26.9. MALDI-MS (positive mode, CHCA): m/z 335.1[M+Na].

(b) The product from (a) above (890 mg, 2.852 mmol) and 10% Pd/C (200mg) in ethyl acetate:methanol (3:2, 20 mL), stirred under H₂ atmosphere(balloon) at room temperature for 8 and then the mixture was filtered,concentrated and purified by flash chromatography (2:8, ethylacetate:petroleum ether) to obtain

(608 mg, 96% yield). R_(f)=0.20 (2:8, ethyl acetate:petroleum ether).[α]_(D) ²⁵=−19.0 (c 1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 7.28-7.19(m, 5H), 4.12 (dt, J=12.5, 8.2, 6.2 Hz, 1H), 3.80 (ddd, J=8.0, 4.7, 3.0Hz, 1H), 3.51 (dd, J=12.0, 3.0 Hz, 1H), 3.28 (dd, J=12.0, 4.7 Hz, 1H),3.04 (dd, J=14.0, 6.5 Hz, 1H), 2.82 (dd, J=14.0, 6.5 Hz, 1H), 1.40 (s,6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 136.9, 129.2, 128.4, 126.6, 108.7,81.1, 77.06, 61.9, 39.3, 27.2, 27.0. MALDI-MS (positive mode, CHCA): m/z345.0 [M+Na]⁺.

(c) The product of (b) above, was converted into the triflate productusing the method mentioned in procedure 1(a) above to give

as a colourless liquid (yield: 78%). [α]_(D) ²⁵=−15.5 (c 1.0, CHCl₃). ¹HNMR (250 MHz, CDCl₃): δ 7.32-7.19 (m, 5H), 4.23-4.08 (m, 2H), 4.01-3.91(m, 2H), 3.16 (dd, J=13.5, 6.0 Hz, 1H), 2.81 (dd, J=13.5, 6.0 Hz, 1H),1.42 (s, 3H), 1.39 (s, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 211.4, 135.8,129.1, 128.8, 127.2, 110.0, 77.7, 76.9, 74.5, 39.2, 27.2, 26.6.

(d) The procedure of 1(a) above was repeated using the product of (c)above to give

which was purified by flash chromatography (1:9, ethyl acetate:petroleumether) as a colourless liquid (yield: 93%). R_(f)=0.42 (15:85, ethylacetate:petroleum ether). [α]_(D) ²⁵=+4.9 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.30-7.22 (m, 5H), 4.13-4.05 (m, 2H), 3.92-3.80 (m, 3H),3.61-3.53 (m, 2H), 3.45-3.34 (m, 2H), 3.03 (dd, J=14.0, 6.7 Hz, 1H),2.88 (dd, J=14.0, 6.7 Hz, 1H), 1.53-1.24 (m, 26H), 1.37 (br. s, 12H),0.86 (t, J=6.5 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 137.3, 129.3,128.3, 127.6, 126.5, 108.9, 108.2, 79.7, 78.2, 77.7, 75.6, 72.7, 71.9,59.8, 39.5, 31.9, 29.67, 29.64, 29.58, 29.54, 29.4, 29.3, 28.1, 27.2,26.9, 26.4, 25.6, 22.6, 14.1. MALDI-MS (positive mode, CHCA): m/z 610.9[M+Na]⁺.

(e) The procedure of 1(b) was repeated using the product of (d) above togive

which was purified by flash chromatography (15:85, ethylacetate:petroleum ether) as a colourless solid (yield: 72%). R_(f)=0.35(2:8, ethyl acetate:petroleum ether). [α]_(D) ²⁵=+9.2 (c 1.0, CHCl₃). ¹HNMR (250 MHz, CDCl₃): δ 7.31-7.23 (m, 5H), 5.70 (d, J=9.0 Hz, 1H),4.19-4.00 (m, 4H), 3.95-3.88 (m, 1H), 3.71 (dd, J=9.7, 3.0 Hz, 1H),3.46-3.33 (m, 3H), 3.03 (dd, J=14.0, 6.5 Hz, 1H), 2.88 (dd, J=14.0, 6.5Hz, 1H), 2.14 (dt, J=10.5, 7.2, 2.7 Hz, 1H), 1.66-1.27 (m, 84H), 0.90(t, J=6.5 Hz, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.3, 137.2, 129.3,128.3, 126.5, 109.0, 107.8, 79.7, 78.4, 77.7, 75.9, 72.1, 48.1, 39.6,36.9, 31.9, 29.6, 29.5, 29.4, 29.35, 29.32, 29.0, 28.0, 27.3, 27.1,26.4, 25.79, 25.72, 22.6, 14.1. MALDI-MS (positive mode, DHB): m/z 962.4[M+Na]. Anal. Calcd for C₆₀H₁₀₉NO₆ (939.83): C, 76.62; H, 11.68; N,1.49. Found: C, 76.70; H, 11.59; N, 1.53.

(f) The procedure of 1(c) was repeated using the product of (e) above togive

as a colourless solid (yield: 70%), hereafter referred to as Compound 6(‘4-Deoxy-4-Phenyl-Threitol-Ceramide Analog’). ¹H NMR (250 MHz, C₅D₅N):δ 8.46 (d, J=8.5 Hz, 1H), 7.37-7.13 (m, 5H), 4.22-4.04 (m, 7H),3.94-3.92 (m, 2H), 3.17 (dd, J=14.0, 4.6 Hz, 1H), 3.03 (dd, J=14.0, 7.5Hz, 1H), 2.31 (br. t, J=7.2 Hz, 2H), 1.73-1.70 (m, 4H), 1.19-1.13 (m,68H), 0.74 (t, J=6.7 Hz, 6H). MALDI-MS (positive mode, DHB): m/z 882.9[M+Na]⁺. Anal. Calcd for C₅₄H₁₀₁NO₆ (859.76): C, 75.38; H, 11.83; N,1.63. Found: C, 75.36; H, 12.03; N, 1.68.Compound 7: 4-Deoxy-4-Phenyl-Threitol-22-(Z)-Ceramide

(a) The procedure of 1(b) was repeated using 22-(Z)-hexacosanoic acidand the product of 6(d) above e.g.

to give

as a colourless solid (yield: 72%). R_(f)=0.38 (2:8, ethylacetate:petroleum ether). [α]_(D) ²⁵=+8.0 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.31-7.22 (m, 5H), 5.66 (d, J=9.2 Hz, 1H), 5.36-5.31 (m,2H), 4.12-3.98 (m, 4H), 3.91-3.84 (m, 1H), 3.67 (dd, J=10.0, 3.5 Hz,1H), 3.43-3.28 (m, 3H), 2.99 (dd, J=14.0, 6.6 Hz, 1H), 2.84 (dd, J=14.0,6.5 Hz, 1H), 2.00 (dt, J=10.5, 7.2, 2.7 Hz, 2H), 2.02-1.97 (m, 4H),1.57-1.23 (m, 76H), 0.91-0.81 (m, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ172.3, 137.2, 130.1, 129.5, 129.3, 128.4, 126.6, 109.0, 107.8, 79.7,78.4, 77.7, 75.9, 72.2, 71.2, 48.1, 39.6, 36.9, 31.9, 29.76, 29.71,29.5, 29.4, 29.36, 29.31, 29.2, 29.0, 28.0, 27.3, 27.2, 27.1, 26.4,25.8, 25.7, 22.8, 22.6, 14.1, 13.8. MALDI-MS (positive mode, DRS): m/z960.1 [M+Na]⁺. Anal. Calcd for C₆₀H₁₀₇NO₆ (937.81): C, 76.79; H, 11.49;N, 1.49. Found: C, 76.86; H, 11.57; N, 1.55.

(b) The procedure of 1(c) was repeated using the product of (a) above togive

as a colourless solid hereafter referred to as Compound 7(‘4-Deoxy-4-Phenyl-Threitol-22-(Z)-Ceramide’) (yield: 68%). ¹H NMR (250MHz, C₅D₅N): δ 8.46 (d, J=8.4 Hz, 1H), 737-7.13 (m, 5H), 5.41-5.25 (m,2H), 4.22-4.05 (m, 7H), 3.94-3.92 (m, 2H), 3.17 (dd, J=14.0, 4.6 Hz,1H), 3.05 (dd, J=14.0, 7.5 Hz, 1H), 2.31 (br. t, J=7.2 Hz, 2H),1.99-1.87 (m, 4H), 1.80-1.64 (m, 4H), 1.17-1.13 (m, 60H), 0.79-0.71 (m,6H). MALDI-MS (positive mode, DHB): m/z 881.1 [M+Na]⁺. Anal. Calcd forC₅₄H₉₉NO₆ (857.75): C, 75.56; H, 11.63; N, 1.63. Found: C, 75.47; H,11.58; N, 1.68.Compound 8: D-Glycerol-phosphate Ceramide

(a) A solution of

(150 mg, 0.204 mmol) in CH₂Cl₂ (4 mL), was prepared as described byMayer, et al., Angew. Chem., 106:2289 (1994); Angew. Chem., Int. Ed.,33:2177 (1994) and Kratzer, et al., Eur. J. Org. Chem., 291 (1998), andcombined with 0.45 M solution of tetrazole in acetonitrile (1.16 mL,0.50 mmol) at room temperature. After being stirred for 10 minutes, asolution of compound

as described by Chen, et al., J. Org. Chem., 63:6511 (1998), (97 mg,0.265 mmol), in dry CH₂Cl₂ (3 mL) was added at the same temperature. Thereaction mixture was stirred for 2.5 hours, and then t-Butylhydroperoxide (0.26 mL, 0.266 mmol) was added to the reaction mixture.The reaction mixture was further stirred for 15 minutes and the mixturewas extracted with CH₂Cl₂ and water, the organic layer was washed withsaturated NaHCO₃ and brine, dried aver anhydrous MgSO₄. The solvent wasevaporated to give crude material, which was purified by flashchromatography (4:6, ethyl acetate:petroleum ether) to give pure

as a colorless solid (191 mg, 92%) mp 87° C. R_(f) 0.40 (6:4, ethylacetate:petroleum ether). [α]_(D) ²⁵=+4.0 (c 1.0, CHCl₃). ¹H NMR (400MHz, CDCl₃, mixture of diastereomers): δ 7.36-7.32 (m, 5H), 6.01 (d,J=8.9 Hz, 1H), 5.89 (d, J=9.3 Hz, 1H), 5.07 (d, J=8.5 Hz, 2H), 5.04 (d,J=8.6 Hz, 2H), 4.29-4.19 (m, 3H), 4.10-3.86 (m, 6H), 3.76-3.69 (m, 1H),2.11-2.03 (m, 2H), 1.55-1.18 (m, 72H), 1.38 (s, 6H), 1.37 (s, 3H), 1.36(s, 3H), 1.31 (s, 6H), 1.28 (s, 3H), 1.27 (s, 3H), 0.85 (t, J=6.6 Hz,61-1). ¹³C NMR (100 MHz, CDCl₃): δ 172.64, 172.61, 135.6, 135.5,128.6-127.7 (in), 109.9, 108.0, 77.5, 75.3, 73.9, 65.9, 48.2, 36.7,31.9, 29.6, 29.5, 29.3, 27.8, 26.5, 25.5, 25.1, 22.6, 14.1. ³¹P NMR (162MHz, CDCl₃): δ 0.1, 0.04. MALDI-MS (positive mode, Matrix CHCA): m/z1043.2 [M+Na]⁺. Anal. Calcd for C₆₀H₁₁₀NO₉P (1019.79): C, 70.62; H,10.86; N, 1.37. Found: C, 70.66; H, 10.94; N, 1.41.

(b) The product of (a) above (160 mg, 0.157 mmol) and 10% Pd/C (50 mg)in methanol (8 mL) was stirred under H₂ atmosphere at room temperaturefor 1 hour. To this reaction mixture triethyl amine (26 μL, 0.188 mmol)was added, and after being stirred for 15 minutes, it was filteredthrough celite and evaporated to give crude material which was submittedto subsequent reaction without any further purification. The resultingcompound

was dissolved in MeOH/CH₂Cl₂ (10:1, 22 mL) containing TFA (150 μL), andwas stirred at room temperature for 3 days. The solvent was evaporatedto give a solid. The solid was filtered and washed thoroughly with ethylacetate and CH₂Cl₂ (to remove soluble organic material). This solid wasdissolved in dioxane (1 mL), a few drops MeOH and triethyl amine whereadded (26 μL, 0.188 mmol) with heating to 60° C. This mixture waslyophilized to give unprotected target molecule

hereinafter referred to as Compound 8 (‘D-Glycerol-phosphate Ceramide’);as a colorless solid (97 mg, 60%). ¹H NMR (250 MHz, C₅D₅N): δ 9.09 (d,J=8.5 Hz, 1H), 5.24-5.09 (m, 2H), 4.96-4.90 (m, 1H), 4.80-4.75 (m, 2H),4.60-4.52 (m, 2H), 4.44-4.40 (m, 1H), 4.28 (br. d, J=5.5 Hz, 2H), 3.10(q, J=7.5 Hz, 6H), 2.60 (t, J=6.7 Hz, 2H), 2.05-1.32 (m, 81H), 0.97-0.95(m, 6H). ¹³C NMR (150.9 MHz, C₅D₅N): δ 173.4, 75.5, 72.67, 72.65, 72.5,68.3, 65.94, 65.92, 65.8, 45.7, 36.8, 33.3, 32.1, 30.3, 30.2, 30.0,29.8, 29.6, 29.2, 6.4, 22.9, 14.2. ³¹P NMR (162 MHz, C₅D₅N): δ 3.0.MALDI-MS (negative mode, Matrix ATT): m/z 929.1 [M-HNEt₃)]⁻. Anal. Calcdfor C₅₉H₁₁₉NO₉P (1031.86): C, 68.70; H, 11.63; N, 2.72. Found: C, 68.75;H, 10.99; N, 2.79.Compound 9: L-Glycerol-phosphate Ceramide

The procedure described in 8(a) above was followed using isomer

to give a crude material which was purified by flash chromatography(4:6, ethyl acetate:petroleum ether) to give pure

as a colorless solid (195 mg, 94%) mp 85° C. R_(f) 0.47 (6:4, ethylacetate:petroleum ether). [α]_(D) ²⁵=2.5 (c 1.0, CHCl₃). ¹H NMR (400MHz, CDCl₃, mixture of diastereomers): δ 7.40-7.30 (m, 5H), 6.05 (d,J=9.1 Hz, 1H), 5.99 (d, J=9.2 Hz, 1H), 4.32-4.20 (m, 3H), 4.13-3.93 (m,6H), 3.75 (br. dd, J=8.8, 5.6 Hz, 1H), 2.20-2.09 (m, 2H), 1.58-1.13 (m,72H), 1.38 (s, 6H), 1.34 (s, 3H), 1.35 (s, 3H), 1.31 (s, 6H), 1.29 (s,3H), 1.25 (s, 3H), 0.93 (1, J=6.6 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ172.64, 172.61, 135.6, 135.5, 128.6-127.7 (m), 109.9, 108.0, 108.0,77.65, 77.63, 75.46, 75.43, 73.9, 73.8, 65.9, 48.2, 36.7, 31.9, 29.6,29.5, 29.3, 28.9, 27.9, 27.8, 26.6, 26.5, 25.5, 25.1, 22.6, 14.1. ³¹PNMR (162 MHz, CDCl₃): δ 0.3, 0.2. MALDI-MS (positive mode, Matrix CHCA):m/z 1043.2 [M+Na]⁺. Anal. Calcd for C₆₀H₁₁₀NO₉P (1019.79): C, 70.62; H,10.86; N, 1.37. Found: C, 70.69; H, 10.98; N, 1.47.

(b) The procedure described for in 8(a) above was used to give

hereinafter referred to as Compound 9 (‘L-Glycerol-phosphate Ceramide’);as a colorless solid (83 mg, 55%). ¹H NMR (250 MHz, C₅D₅N): δ 9.09 (d,J=8.4 Hz, 1H), 4.87-4.65 (m, 5H), 4.59-4.33 (m, 3H), 4.28 (br. d, J=5.0Hz, 2H), 3.10 (q, J=7.5 Hz, 6H), 2.63 (t, J=6.7 Hz, 2H), 2.05-1.37 (m,81H), 0.99-0.96 (m, 6H). ¹³C NMR (150.9 MHz, C₅D₅N): δ 173.4, 75.6,72.65, 72.5, 68.4, 65.96, 65.7, 46.0, 36.9, 33.4, 32.1, 31.6, 31.2,31.0, 29.8, 29.6, 29.2, 26.4, 22.9, 14.2. ³¹P NMR (162 MHz, C₅D₅N): δ3.6. MALDI-MS (negative mode, Matrix ATT): m/z 929.1 [M-HNEt₃)]⁻. Anal.Calcd for C₅₉H₁₁₉NO₉P (1031.86): C, 68.70; H, 11.63; N, 2.72. Found: C,68.79; H, 10.95; N, 2.78.Compound 10: Inositol ceramide

(a) A stirred solution of (366 mg, 0.691 mmol) of compound

obtained in accordance with well known procedures, see Mayer, et al.,Liebigs Ann./Recueil, 859 (1997) and Jiang, et al., J. Carbohydr. Chem.,6:319 (1987), was mixed, with 2,6-di-tert-butylpyridine (159 mg, 0.83mmol) in anhydrous CH₂Cl₂ (3 ml) and Tf₂O (136 μl, 0.83 mmol) which wasdissolved in 2 ml of CH₂Cl₂, at 0° C. The reaction mixture was stirredat the same temperature for 3 hours. The reaction mixture was taken inethyl acetate and washed with cold water (2×25 mL). The combined organiclayers were washed with brine, dried and evaporated to get the crudeproduct which was purified by flash chromatography (8:92 ethylacetate:petroleum ether containing drops of Et₃N) to give (413 mg, 90%)of

Rf 0.41 (1:9, ethyl acetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ7.41-7.28 (m, 15H), 4.88 (d, J=11.9 Hz, 1H), 4.78-4.67 (m, 5H), 4.30(dd, J=6.0, 3.7 Hz, 1H), 4.17 (t, J=6.4 Hz, 1H), 4.03 (t, J=7.6 Hz, 1H),3.97 (dd, J=9.1, 6.5 Hz, 1H), 3.77 (dd, J=8.0, 3.6 Hz, 1H), 1.83-1.30(m, 10H). ¹³C NMR (62.5 MHz, CDCl₃): δ 137.6, 137.32, 137.29, 128.4,128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 111.1, 87.7, 79.0, 77.83,77.77, 76.4, 74.6, 73.6, 73.3, 37.0, 34.5, 25.0, 23.9, 23.6.

(b) The procedure of 1(a) above was repeated using a solution of NaH(60%, 7 mg, 0.181 mmol), 58 mg (0.151 mmol) of sphingosine componenti.e.

in 1 ml of anhydrous DMF. After thirty minutes of stirring, a solutionof the product of (a) above, (100 mg, 0.151 mmol) in anhydrous DMF (1.5ml), was added, at this same temperature. After stirring overnight, thereaction mixture was quenched by adding an aqueous solution of NH₄Cl. Itwas taken in EtOAc and the layers were separated. The organic layer waswashed with water, dried over anhydrous MgSO₄ and evaporated to dryness.The crude material was purified by flash chromatography (1:9, ethylacetate:petroleum ether) to give the pure compound

as a colorless liquid (108 mg, 80%), R_(f)=0.44 (1:9, ethylacetate:petroleum ether). ¹H NMR (250 MHz, CDCl₃): δ 7.45-7.23 (m, 15H),4.88-4.63 (m, 6H), 4.30-3.83 (m, 8H, 3.75-3.69 (m, 1H), 3.56-3.47 (m,1H), 3.40-3.35 (m, 1H), 1.72-1.26 (m, 36H), 1.37 (s, 3H), 1.35 (s, 3H),0.88 (t, J=7.2 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 139.0, 138.8,138.7, 138.6, 138.4, 138.3, 128.3, 128.22, 128.18, 128.1, 128.0, 127.59,127.55, 127.5, 127.4, 127.3, 109.7, 108.0, 79.2, 79.1, 78.9, 78.8, 78.7,78.0, 77.9, 77.8, 77.2, 76.25, 76.20, 75.4, 75.3, 74.2, 73.9, 73.7,73.4, 73.3, 73.13, 73.09, 71.7, 71.5, 60.22, 60.16, 37.9, 35.3, 31.9,29.64, 29.61, 29.5, 29.3, 28.13, 28.10, 26.4, 25.7, 25.1, 24.0, 23.5,22.6, 14.0. MALDI-MS (positive mode, DHB): m/z 919.8 [M Na]⁺. Anal.Calcd for C₅₄H₇₇N₃O₈ (896.20): C, 72.37; H, 8.66; N, 4.69. Found: C,71.91; H, 8.36; N, 4.68.

(c) The product of (b) above 160 mg (0.178 mmol), and a pinch ofPd(OH)₂/C in MeOH/CH₂Cl₂/H₂O (7.5:7.5:1, 3 mL) was stirred under H₂atmosphere at room temperature overnight. Then the mixture was filtered,concentrated and co-evaporated with toluene. The resulting syrup wasdissolved in dry DMF (4 mL). Hexacosonoic acid (71 mg, 0.178 mmol),N-hydroxybenzotriazole (24 mg, 0.178 mmol) and1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (34 mg,0.178 mmol) were added successively and the resulting mixture wasstirred at 45° C. for 1 day. Then it was taken in ethyl acetate washedwith water, saturated brine solution, dried over anhydrous MgSO₄, andconcentrated. The residue was purified by flash column chromatography(7:3, ethyl acetate:petroleum ether) to give the compound

(75 mg, 43%). R_(f)=0.23 (7.5:2.5, ethyl acetate:petroleum ether). ¹HNMR (250 MHz, CDCl₁): δ 6.09 (t, J=9.5 Hz, 1H), 4.47-4.40 (m, 1H),4.27-3.68 (m, 10H), 2.36-111 (m, 2H), 1.72-1.18 (m, 82H), 1.44 (s, 3H),1.34 (s, 3H), 0.88 (t, J=6.4 Hz, 6H). MALDI-MS (positive mode, DHB):1002.0 [M+Na]⁺. Anal. Calcd for C₅₉H₁₁₁NO₉ (978.51): C, 72.42; H, 11.43;N, 1.43. Found: C, 72.16; H, 11.46; N, 1.36.

(d) A solution of the product (c) above (46 mg, 0.047 mmol), inMeOH/CH₂Cl₂ (1:1, 2 ml), with a few crystals of CSA were stirred, atroom temperature, for 2 days. The solid thrown out was dried to give 22mg of the compound

hereinafter referred to as Compound 10 (‘inositol-ceramide’); (55%yield). ¹H NMR (250 MHz, C₅D₅N): δ 5.20-4.15 (m, 11H), 2.49-2.38 (m,2H), 2.38-1.10 (m, 72H), 0.91-0.80 (m, 6H). MALDI-MS (positive mode,DHB): m/z 881.6 [M+Na]⁺. Anal. Calcd for C₅₀H₉₉NO₉ (858.32): C, 69.97;H, 11.63; N, 1.63. Found: C, 70.07; H, 11.70; N, 1.67.Compound 11: Inositol ceramide C₁₅ acyl

(a) The product of 10(b) (114 mg (0.127 mmol) e.g.

was combined with 10% Pd/C (100 ng) in MeOH/CH₂Cl₂/H₂O (7.5:7.5:1, 3 mL)and stirred under an H₂ atmosphere at room temperature overnight. Thenthe mixture was filtered, concentrated and co-evaporated with toluene.The resulting syrup was dissolved in dry DMF (4 mL). Palmitic acid (33mg, 0.127 mmol), N-hydroxybenzotriazole (17 mg, 0.127 mmol) and1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (24 mg,0.127 mmol) were added successively and the resulting mixture wasstirred at 45° C. for 1 day. Then it was taken in ethyl acetate washedwith water, saturated brine solution, dried over anhydrous MgSO₄, andconcentrated. The residue was purified by flash chromatography (7:3,ethyl acetate:petroleum ether) to give

(48 mg, 45%). Rf=0.24 (3:4, ethyl acetate:petroleum ether). ¹H NMR (250MHz, CDCl₃): δ 6.05 (t, J=9.3 Hz, 1H), 4.47-4.39 (m, 1H), 4.27-3.67 (m,10H), 2.28-2.13 (, 2H), 1.72-1.20 (m, 62H), 1.44 (s, 3H), 1.34 (s, 3H),0.88 (t, J=6.4 Hz, 6H). MALDI-MS (positive mode, DHB): 861.7 [M+Na]⁺.Anal. Calcd for C₄₉H₉₁NO₉ (838.24): C, 70.11; H, 10.94; N, 1.67. Found:C, 70.19; H, 11.03; N, 1.69.

(b) The product of (a) above (44 mg, 0.047 mmol) in MeOH:CH₂Cl₂ (1:12ml) containing a few crystals of CSA was stirred at room temperature for36 hours. The solid thrown out was filtered and dried to give

hereinafter referred to as Compound 11 (‘inositol-ceramide C₁₅ acyl’);(19 mg, 50%). ¹H NMR (250 MHz, C₅D₅N): δ 5.35-4.25 (m, 11H), 2.48-2.37(m, 2H), 2.37-1.10 (m, 52H), 0.90-0.78 (m, 6H). MALDI-MS (positive mode,DHB): 741.2 [M+Na]⁺. Anal. Calcd for C₄₀H₇₉NO₉ (718.05): C, 66.91; H,11.09; N, 1.95. Found: C, 66.98; H, 11.47; N, 1.39.Compound 12: D-myo-Inositol Ceramide

(a) Compound

was prepared with well known procedures, see Stadelmaier, et al.,Carbohydr. Res., 338:2557 (2003). The above compound (2.66 g, 6.820mmol) in dry toluene (25 mL), was treated with NaH (95% in mineral oil,200 mg, 8.333 mmol), benzyl bromide (900 μL, 7.578 mmol). The reactionmixture was refluxed for 10 hours. The reaction mixture was cooled roomtemperature, and diluted with ethyl acetate, washed with water, driedover MgSO₄ and evaporated to give residue, which was purified by flashchromatography (15:85, ethyl acetate:petroleum ether) from a mixture ofisomers (˜1:1.4) (75% yield) to give

R_(f)=0.32 (15:85, ethyl acetate:petroleum ether). ¹H NMR (600 MHz,CDCl₃): δ 7.37-7.28 (m, 10H), 5.96-5.90 (m, 1H), 5.29 (dd, J=17.4, 1.8Hz, 1H), 5.18 (dd, J 10.8, 1.8 Hz, 1H), 4.87 (d, J=11.4 Hz, 1H,benzyl-H)), 4.76-4.71 (m, 3H, benzyl-H), 4.38-4.36 (m, 1H, allyl-H),4.29 (dd, J=6.0, 4.2 Hz, 1H, H-4), 4.23-4.21 (m, 1H, allyl-H), 4.01 (dd,J=7.2, 6.0 Hz, 1H, H-5), 3.79 (t, =8.1 Hz, 1H, H-2), 3.68 (dd, J=8.1,3.9 Hz, 1H, H-3), 3.59 (dd, J=9.6, 7.2 Hz, 1H, H-6), 3.44 (dd, J=9.6,8.1 Hz, 1H, H-1), 2.32 (br. s, 1H, —OH), 1.80-1.41 (m, 10H). ¹³C NMR(150 MHz, CDCl₃): δ 138.5, 138.1, 135.0, 128.59, 128.57, 128.47, 128.43,128.0, 127.98, 127.94, 127.8, 127.7, 117.2, 110.4, 81.4 (C-6), 80.6(C-2), 78.4 (C-5), 77.4 (C-3), 74.6, 74.0 (C-4), 73.3 (C-1), 72.8, 72.2,37.3, 35.0, 25.0, 23.9, 23.6. MALDI-MS (positive mode, DHB): m/z, 503.5[M Na]⁺.

(b) The procedures of 1(a) and 1(b) were repeated using the product of(a) above to give

which was purified by flash chromatography (5:95, ethylacetate:petroleum ether), (yield: 87%). R_(f)=0.54 (1:9, ethylacetate:petroleum ether). [α]_(D) ²⁵=+1.4 (c 1.0, CHCl₃). ¹H NMR (600MHz, CDCl₃): δ 7.39-7.23 (m, 20H), 5.96-5.90 (m, 1H), 5.20 (dd, J=17.4,1.8, Hz, 1H), 5.10 (dd, J=10.8, 1.8 Hz, 111), 4.84-4.81 (m, 2H,benzyl-H), 4.72 (d, J=12.0 Hz, 1H, benzyl-H), 4.68-4.64 (m, 2H,benzyl-H), 4.59-4.54 (m, 2H, benzyl-H), 4.46 (d, J=11.4 Hz, 1H,benzyl-H), 4.26 (t, J=4.8 Hz, 1H, H-4), 4.19-4.16 (m, 3H, H-5, H-3,allyl-H), 4.14-4.06 (m, 2H, H-1′, allyl-H), 3.99-3.96 (m, 1H, H-1′),3.81 (d, J=1.8 Hz, 1H, H-1), 3.75 (dd, J=9.6, 1.8 Hz, 1H, H-2),3.72-3.70 (m, 1H, H-2), 3.58-3.56 (m, 2H, H-3′, H-4′), 3.33 (dd, J=8.4,2.4 Hz, 1H, H-6), 1.61-1.41 (m, 10H), 1.26-1.24 (m, 26H), 0.88 (t, J=7.2Hz, 1H, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 138.5-127.4 (m, 24C), 135.1,116.8, 109.7, 79.6 (C-4′), 79.1 (C-1, C-6), 78.8 (C-2), 78.7 (C-3′),78.0 (C-5), 76.3 (C-3), 73.8 (C-4), 73.6, 73.5 (C-1′), 73.3, 71.9, 70.9,62.4 (C-2′), 37.5, 35.0, 25.0, 23.9, 23.6, 22.7, 14.1. MALDI-MS(positive mode, DHB): m/z 1009.1 [M+Na]⁺. Anal. Calcd for C₆₁H₈₃N₃O₈(985.62): C, 74.28; H, 8.48; N, 4.26. Found: C, 74.19; H, 8.42; N, 4.28.

(c) The product of (b) above (700 mg, 0.710 mmol) in a mixture oftoluene/ethanol/1 M aq. HCl (3:6:1, 15 mL) was heated at 60° C. for 3hours. The solvent was co-evaporated with toluene, dried in vacuo, theresidue was dissolved in dry DMF (10 mL), NaH (60% mineral oil, 85 mg,3.553 mmol) was added and stirred for 30 minutes, at room temperature,and then benzyl bromide (220 μL, 1.77 mmol) was added. The reactionmixture was stirred another 5 hours, and then extracted with diethylether (2×20 mL), the ether layer was washed with and water, dried overMgSO₄ and evaporated to give crude material, which was purified by flashchromatography (5:95, ethyl acetate:petroleum ether) to give

(655 mg, 85% yield). R_(f)=0.40 (5:95, ethyl acetate:petroleum ether).[α]_(D) ²⁵=+2.8 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.44-7.28 (m,30H), 5.96-5.90 (m, 1H), 5.32 (dd, J=17.4, 1.8 Hz, 1H), 5.14 (dd,J=10.8, 1.8 Hz, 1H), 4.88-4.62 (m, 11H), 4.54 (d, J=11.4 Hz, 1H),4.29-4.27 (m, 1H), 4.22-4.12 (m, 3H), 4.08 (br. s, 1H), 3.96 (br. s,3H), 3.87-3.83 (m, 3H), 3.68-3.66 (m, 2H), 1.49-1.32 (m, 26H), 0.94 (t,J=7.2 Hz, 3H). ¹³C NMR (1.50 MHz, CDCl₃): δ 138.5-127.2 (m, 36C), 135.4,116.2, 79.8, 79.2, 79.0, 78.9, 78.7, 78.38, 78.36, 76.0, 74.6, 74.4,73.8, 73.17, 73.15, 73.0, 72.18, 72.10, 62.5, 32.0, 29.9, 29.89, 29.80,29.79, 29.76, 29.4, 27.0, 25.7, 22.7, 14.2. MALDI-MS (positive mode,DHB): m/z 1110.4 [M+Na]⁺. Anal. Calcd for C₆₉H₅₇N₃O₈ (1085.65): C,76.28; H, 8.07; N, 3.87. Found: C, 76.36; H, 8.17; N, 3.95.

(d) The product of (c) above (650 mg, 0.598 mmol) in diethyl ether (5mL) was added to a suspension of LiAlH₄ (46 mg, 1.210 mmol) in diethylether (10 mL) at 0° C. dropwise. The reaction mixture was slowly broughtto the room temperature and refluxed for 1 hour. The reaction mixturewas quenched with methanol and extracted with ethyl acetate (2×15 mL)and Water. The organic layer was washed with brine and dried over MgSO₄.Removal of the solvent gave a crude amine. The procedure in 1(b) abovewas repeated for the coupling of this amine to the carboxylic acid togive

(yield: 65%). R_(f)=0.57 (15:85, ethyl acetate:petroleum ether). [α]_(D)²⁵=+1.6 (c 1.0, CHCl₃). NMR (600 MHz, CDCl₃): δ 7.37-7.17 (m, 30H), 7.04(d, J=8.6 Hz, 1H), 5.96-5.91 (m, 1H), 5.27 (dd, J=17.4, 1.8 Hz, 1H),5.15 (dd, J=10.8, 1.8 Hz, 1H), 4.83-4.43 (m, 13H, benzyl-H, H-1′),4.33-4.31 (m, 1H), 4.23-4.21 (m, 1H), 4.19-4.02 (m, 2H, H-4, H-2′), 3.91(dd, J=9.6, 2.4 Hz, H-2), 3.88-382 (m, 3H, H-3′, H-6, H-1), 3.73 (dd,=9.6, 3.0 Hz, 1H, H-3), 3.70 (dd, J=10.2, 2.4 Hz, 1H, H-5), 3.66-3.62(m, 1H, H-1′), 3.48-3.44 (m, 1H, H-4′), 2.21-2.17 (m, 2H), 1.57-1.27 (m,72H), 0.91 (t, J=7.2 Hz, 6H). ¹³C NMR (150 MHz, CDCl₃): δ 172.8,139.2-1272 (m, 36C), 135.1, 117.1, 80.8 (C-4′), 80.0 (C-1), 79.6 (C-3),79.44 (C-2), 79.41, 79.3 (C-5, C-6), 78.2 (C-3′), 75.3 (C-4), 74.6,74.4, 73.8, 73.8 (C-1′), 73.6, 73.1, 72.8, 72.7, 71.6, 51.0 (C-2′),36.8, 32.0, 29.9, 29.79, 29.77, 29.72, 29.6, 29.44, 29.43, 26.4, 25.9,25.8, 22.7, 14.1. MALDI-MS (positive mode, DHB): m/z 1464.2 [M+Na]⁺.Anal. Calcd for C₉₅H₁₃₉NO₉ (1438.04): C, 79.29; 9.74; N, 0.97. Found: C,79.35; H, 9.81; N, 1.09.

(e) The compound of (d) above (650 mg, 0.452 mmol) as a solution inethanol (15 mL), was treated with DBU (10 μL, 0.065 mmol), andtris(triphenylphospine) ruthenium (H) chloride (130 mg, 0.135 mmol). Thereaction mixture was heated to reflux at 90° C. for 30 minutes. Thesolvent was evaporated to give isomerized product (R_(f)=0.54, 15:85,ethyl acetate:petroleum ether), which was dissolved in 1 M aq. HCl inacetone (1:9, 15 ml) and the reaction mixture was heated to reflux at70° C. for 15 minutes. The mixture was cooled to room temperature,neutralized with Et₃N and extracted with ethyl acetate (2×20 mL), theorganic phase was washed with water and brine, dried over MgSO₄ andevaporated to give crude material. Purified by flash chromatography(18:82, ethyl acetate:petroleum ether) to give

(655 mg, 78% yield). R_(f)=0.2 (15:85, ethyl acetate:petroleum ether).[α]_(D) ²⁵=−6.4 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.37-7.23 (m,30H), 7.16 (d, J=8.7 Hz, 1H), 4.88 (d, J=12.0 Hz, 1H), 4.78-4.68 (m,5H), 4.59-4.54 (m, 4H), 4.47-4.42 (m, 3H), 4.09-4.06 (m, 3H), 3.95-3.93(m, 2H), 3.76-3.3.73 (m, 2H), 3.66 (br. d, J=8.4 Hz, 1H), 3.51-3.46 (m,2H), 1.97-2.14 (m, 2H), 1.52-1.24 (m, 72H), 0.89 (t, J=7.2 Hz, 6H). ¹³CNMR (150 MHz, CDCl₃): δ 172.9, 138.9-127.3 (m, 36C), 80.7, 80.3, 79.9,79.3, 79.1, 74.4, 74.2, 74.0, 73.9, 73.5, 73.3, 72.0, 71.7, 70.2, 51.9,36.9, 31.9, 29.9, 29.77, 29.75, 29.70, 29.5, 29.42, 29.4, 26.3, 25.8,22.7, 14.1. MALDI-MS (positive mode, DHB): m/z 1424.7 [M+Na]⁺. Anal.Calcd for C₉₂H₁₃₅NO₉ (1398.01): C, 78.98; H, 9.73; N, 1.00. Found: C,79.08; H, 9.84; N, 1.13.

(f) The procedure of 6(a) for reducing an OH group was repeated usingthe product of (e) above to give

which was purified by flash chromatography (2:98 ethyl acetate:toluene)as colourless solid (60%). R_(f)=0.57 (4:96, ethyl acetate:toluene).[α]_(D) ²⁵=+11.2 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.38-7.17(m, 30H), 6.36 (d, J=8.6 Hz, 1H, —NH), 4.83-4.36 (m, 12H, benzyl-H),4.18-4.14 (m, 2H, H-2′, H-4), 4.00 (br. d, J=9.0 Hz, 1H, H-1′), 3.93(dd, J=10.0, 3.0 Hz, 1H), H-2), 3.84 (t, J=4.0 Hz, 1H, H-3′), 3.78 (d,J=3.0 Hz, 1H, H-1), 3.76 (br. t, J=9.0 Hz, 1H, H-5), 3.72 (dd, J=10.0,2.0 Hz, 1H, H-3), 3.66 (br. d, J=9.0 Hz, 1H, H-1′), 3.51 (ddd, J=12.0,8.0, 4.0 Hz, 1H, H-4′), 1.98-1.94 (m, 1H, H-6), 1.82-1.72 (m, 2H),1.63-1.61 (m, 1H, H-6), 1.43-1.24 (m, 72H), 0.86 (t, J=6.5 Hz, 6H). ¹³CNMR (150 MHz, CDCl₃): δ 173.3, 139-127.2 (m, 36C), 80.2 (C-2, C-3′),79.8 (C-3, C-4′), 75.9 (C-4), 75.8 (C-1), 75.1 (C-5), 74.0, 73.8, 72.5,71.9, 71.0 (C-1), 51.3 (C-2′), 36.6, 31.9, 30.5, 30.2, 29.8, 29.75,29.73, 29.69, 29.65, 29.5, 29.4, 29.3, 26.0, 25.7, 22.7, 14.1. MALDI-MS(positive mode, DHB): m/z 1408.6 [M+Na]⁺. Anal. Calcd for C₉₂H₁₃₅NO₈(1382.02): C, 79.89; H, 9.84; N, 1.01. Found: C, 79.98; H, 9.99; N,1.18.

(g) The product of (f) above (150 mg, 0.108 mmol) and 20% Pd(OH)₂/C (150mg) in MeOH/CH₂Cl₂/H₂O (7.5:7.5:1, 6 mL) was stirred under H₂ atmosphereat room temperature for 2 hours. The product precipitated and wasdissolved by the addition of a mixture of solventsmethanol/CH₂Cl₂/petroleum ether and with warming. After filtration, thefiltrate was concentrated to give colourless solid

hereafter referred to as Compound 12 (‘D-myo-inositol Ceramide Analog’)(86 mg, 95% yield). ¹H NMR (250 MHz, C₅D₅N): δ 8.45 (d, J=8.5 Hz, 1H),5.16 (dd, J=8.0, 4.2 Hz, 1H), 4.58-4.43 (m, 4H), 4.33-4.22 (m, 5H),2.45-2.30 (m, 3H), 1.91-1.77 (m, 3H), 1.28-1.21 (m, 70H), 0.83 (t, J=6.7Hz, 6H). MALDI-MS (positive mode, DHB): m/z 864.4 [M+Na]⁺. Anal. Calcdfor C₅₀H₉₉NO₈ (841.74): C, 71.30; H, 11.85; N, 1.66. Found: C, 71.41; H,11.98; N, 1.73.Compound 13: D-myo-Inositol Ceramide Salt

(a) To stirred solution of the product of 12(e) above e.g.

(200 mg, 0.143 mmol) in DMF/THF (1:1, 2 mL), SO₃.NMe₃-complex (40 mg,0.287 mind) was added, and the reaction mixture was stirred for 24 hoursat room temperature. After completion of reaction, it was extracted withCH₂Cl₂ (2×10 mL) and the organic phase was dried and concentrated, whichwas purified by flash chromatography to give colourless solid. Which wasdissolved in MeOH/CH₂Cl, (1:1, 2 mL) and passed through the Dowex 50×8H⁺ion exchange resin (Na⁺-form) and eluted with mixture of MeOH/CH₂C19(1:1, 100 mL). The solvent was evaporated to obtain solid,

which was purified by flash chromatography to give a colourless solid(203 mg, 95% yield). This compound debenzylated using similar procedureas described for compound 12(f) above, to give a colourless solid

Hereafter referred to as Compound 13 (‘D-myo-Inositol Ceramide’) (yield:40% yield). ¹H NMR (400 MHz, C₅D₅N: CD₃OD): δ 8.50 (d, J=8.5 Hz, 1H),5.10 (dd, J=10.0, 2.0 Hz, 1H), 4.58-4.55 (m, 2H), 4.48-4.42 (m, 2H),4.26-4.17 (m, 3H), 4.06-3.99 (m, 2H), 3.91-3.88 (m, 1H), 2.31-2.26 (m,2H), 1.63-1.10 (m, 72H), 0.90 (t, J=5.7 Hz, 6H). MALDI-MS (negativemode, ATT): m/z 938.7 [M−Na]⁻. Anal. Calcd for C₅₀H₉₈NNaO₁₂S (959.67):C, 62.53; H, 10.29; N, 1.46. Found: C, 62.59; H, 10.38; N, 1.51.Compound 14: 4-(S)-Phenyl Threitol Ceramides

(a) L-(+)-tartaric acid was converted to a corresponding triflate inaccordance with well known procedures, see Wagner, et al., J. Chem. Soc.Perkins Trans., 1, 780 (2001); Su, et al., Tetrahedron., 57 2147 (2001);Surivet, et al., Tetrahedron Lett., 39:7249 (1998) and Surivet, et al.,Tetrahedron., 55:1311 (1999), all of which are incorporated byreference. The synthesis of the triflate results in a diastereomericmixture. This mixture is separated, following the references citedsupra, to yield two compounds.

(b) The procedure of 1(a) above was repeated with the triflate from (a)above, where R₁=OTBDPS and R₂═H e.g.

to give

which was purified via flash chromatography to give as a colorlessliquid, in a yield of 84%. R_(f)=0.62 (1:9, ethyl acetate:petroleumether). [α]_(D) ²⁵=+20.5 (c 1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ7.71-7.17 (m, 15H), 4.90 (d, J=4.5 Hz, 1H), 4.27-4.20 (m, 1H), 4.15-4.06(m, 1H), 3.95 (dd, J=8.0, 4.5 Hz, 1H), 3.79-3.70 (m, 2H), 3.52 (dt,J=8.7, 2.0 Hz, 1H), 3.40 (t, J=9.0 Hz, 1H), 3.28-3.16 (m, 2H), 1.54-1.20(m. 26H), 1.42 (s, 3H), 1.37 (s, 3H), 1.32 (s, 3H), 1.28 (s, 3H), 1.06(s, 9H), 0.88 (t, J=6.5 Hz, 3H). ¹³C NMR (62.5 MHz, CDCl₃): δ 140.4,136.07, 136.02, 133.5, 133.2, 129.5, 129.4, 127.7, 127.4, 127.3, 127.2,127.0, 109.2, 108.1, 81.1, 77.7, 76.8, 75.7, 75.5, 72.8, 72.6, 59.7,31.9, 29.6, 29.59, 29.53, 29.4, 29.3, 28.0, 27.0, 26.88, 26.82, 26.4,25.6, 22.6, 19.3, 14.1. MALDI-MS (positive mode, CHCA): m/z 865.2 [MNa]⁺. Anal. Calcd for C₅₀H₇₅N₃O₆Si (842. 23): C, 71.30; H, 8.98; N,4.99. Found: C, 71.39; H, 9.03; N, 5.07.

(c) The procedure of 1(b) above was repeated using the product of (b)above to give

which was purified via flash chromatography (8:9.2 ethylacetate:petroleum either), as a colorless solid, in 72% yield.R_(f)=0.52 (1:9, ethyl acetate:petroleum ether). [α]_(D) ²⁵=17.9 (c 1.0,CHCl₃). NMR (250 MHz, CDCl₃): δ 7.69-7.16 (m, 15H), 5.72 (d, J=8.7 Hz,1H), 4.84 (d, J=4.7 Hz, 1H); 4.19-4.08 (On, 4H), 3.87 (dd, J=7.6, 4.5Hz, 1H), 3.59 (br. d, J=9.2 Hz, 1H), 3.33 (br, d, =10.5 Hz, 1H), 3.17(br. d, J=4.5 Hz, 2H), 2.12-2.06 (m, 2H), 1.56-1.25 (m, 72H), 1.40 (s,3H), 1.31 (s, 3H), 1.30 (s, 3H), 1.19 (s, 3H), 1.05 (s, 9H), 0.89 (t,J=6.7 Hz, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 170.4, 139.5-127.1 (in),109.1, 108.1, 80.3, 77.6, 77.1, 75.7, 72.6, 71.7, 65.2, 58.9, 31.8,29.6, 29.5, 29.3, 29.2, 28.0, 26.9, 26.6, 26.5, 26.3, 25.5, 22.6, 20.6,14.0. MALDI-MS (positive mode, DHB): m/z 1217.7 [M+Na]⁺. Anal. Calcd forC₇₆H₂₇NO₇Si (1194. 90): C, 76.39; H, 10.71; N, 1.17. Found: C, 76.45; H,10.81; N, 1.21.

(d) The product of (c) above (200 mg, 0.169 mmol) in THF and 1.0Msolution of TBAF (0.2 ml, 0.203 mmol), was stirred at room temperaturefor 24 hours. The reaction mixture was then taken up in ethyl acetate,washed with water, then a saturated brine solution, and then dried overanhydrous MgSO₄, and concentrated. The residue was purified, via flashchromatography, to give

as a colorless solid (132 mg, 82% yield). R_(f)=0.42 (4:6, ethylacetate:petroleum ether). J=6.7 (c 0.5, CHCl₃). ¹H NMR (250 MHz, CDCl₃):δ 7.41-7.32 (m, 5H), 5.68 (d, J=9.0 Hz, 1H), 4.87 (d, J=5.5 Hz, 1H),4.32-3.96 (m, 5H), 3.60 (dd, J=10.0, 3.8 Hz, 1H), 3.39 (dd, J=10.0, 2.5Hz, 1H), 3.24 (dd, J=10.5, 5.5 Hz, 1H), 3.14 (dd, J=10.5, 4.0 Hz, 1H),2.12 (dt, J=7.5, 3.5 Hz, 2H), 1.54-1.25 (m, 72H), 1.43 (s, 3H); 1.41 (s,3H), 1.38 (s, 3H), 1.33 (s, 3H), 0.88 (t, J=6.7 Hz), 6H). ¹³C NMR (62.5MHz, CDCl₃): δ 172.5, 139.5, 128.3, 127.8, 126.1, 109.3, 107.9, 81.0,77.6, 76.1, 73.0, 72.1, 71.2, 48.1, 36.9, 31.9, 29.6, 29.5, 29.3, 29.0,27.05, 27.01, 26.4, 25.7, 22.6, 14.1. MALDI-MS (positive mode, CHCA):m/z 979.4 [M+Na]⁺. Anal. Calcd for C₆₀H₁₀₉NO₇ (956.51): C, 75.34; H,11.49; N, 1.46. Found: C, 75.40; H, 11.55; N, 1.51

(e) The procedures of 1(c) above was repeated using the product of (d)above, yielding a colorless solid,

hereafter referred to as Compound 14 (‘4-(S)-Phenyl Threitol Ceramide’),at a yield of 68%. ¹H NMR (600 MHz, C₅D₅N): δ 8.52 (d, J=8.5 Hz, 1H),7.79-7.27 (m, 5H), 5.39 (d, J=7.8 Hz, 1H), 5.18-4.94 (m, 1H), 4.93-4.91(m, 1H), 4.30-4.19 (m, 5H), 4.12-4.08 (m, 2H), 2.41 (br. t, J=7.5 Hz,2H), 1.92-1.25 (m, 72H), 0.87 (t, J=6.5 Hz, 6H). ¹³C NMR (150.9 MHz,C₅D₅N): δ 173.3, 114.4, 128.3, 128.1, 127.2, 76.2, 75.4, 75.2, 74.6,72.8, 71.3, 70.0, 51.8, 36.8, 32.1, 30.3, 30.0, 29.7, 29.6, 26.6, 26.4,22.9, 14.3. MALDI-MS (positive mode, CHA): m/z 899.1 [M+Na]⁺. Anal.Calcd for C₅₄H₁₀₁NO₇ (876.38): C, 74.01; H, 11.62; N, 1.60. Found: C,73.98; H, 11.59; N, 1.62.Compound 15: 4-(R)-Phenyl Threitol Ceramide

(a) The procedure of 1(a) above was repeated using the product of 14(a)above, where R₁═H and R₂=OTBDPS e.g.

to give

which was purified by flash chromatography (7:9.3 ethylacetate/petroleum ether), as a colorless liquid, in a yield of 86%.R_(f)=0.59 (1:9, ethyl acetate:petroleum ether). [α]_(D) ²⁵=−15.0 (c1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 7.76-7.14 (m, 15H), 4.82 (d,J=6.5 Hz, 1H), 4.21-4.06 (m, 3H), 3.86 (dd, J=9.5, 5.5 Hz, 1H),3.87-3.68 (m, 3H), 3.51 (dt, J=9.5, 2.2 Hz, 1H), 3.34 (dd, J=1.0.0, 8.5Hz, 1H), 1.58-1.25 (m, 26H), 1.41 (s, 3H), 1.36 (s, 3H), 1.33 (s, 3H),1.31 (s, 3H), 1.04 (s, 9H), 0.87 (t, J=6.7 Hz), ¹³C NMR (62.5 MHz,CDCl₃): δ 139.5, 136.1, 135.9, 133.6, 133.3, 129.4, 129.3, 127.88,127.80, 127.4, 127.2, 127.1, 109.1, 108.1, 80.3, 77.7, 77.5, 77.1, 75.7,72.6, 71.7, 59.7, 31.8, 29.6, 29.5, 29.55, 29.50, 29.47, 29.41, 29.3,29.2, 28.0, 27.0, 26.6, 26.4, 26.3, 25.5, 25.4, 22.6, 19.3, 14.1.MALDI-MS (positive mode, CHCA): m/z 865.4 [M+Na]⁺. Anal. Calcd forC₅₀H₇₅N₃O₆Si (842. 23): C, 71.30; H, 8.98; N, 4.99. Found: C, 71.42; H,9.07; N, 5.04.

(b) The procedures of 14(b) above was repeated but using the product of(a) above to give

which was purified by flash chromatography (1:9 ethyl acetate/petroleumether) the pure compound was obtained as a colorless solid in a yield of69%. R_(f)=0.49 (1:9, ethyl acetate:petroleum ether). [α]_(D) ²⁵=−5.9 (c1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 7.77-7.18 (m, 15H), 5.88 (d,J=9.0 Hz, 1H), 4.74 (d, J=6.5 Hz, 1H), 4.10-4.07 (m, 3H), 3.97 (dd,J=8.0, 6.5 Hz, 1H), 3.77 (dt, 5.7, 2.2 Hz, 1H), 3.55 (br. d, J=9.0 Hz,1H), 3.27 (br. d, J=8.5 Hz, 1H); 3.00-2.97 (m, 2H), 2.16-2.08 (m, 2H),1.58-1.25 (M, 72H), 1.39 (s, 3H), 1.33 (s, 3H), 1.29 (s, 3H), 1.18 (s,3H), 1.04 (s, 9H), 0.88 (t, J=6.5 Hz, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ172.6, 139.6, 136.1, 135.9, 133.6, 133.2, 129.4, 127.9, 127.4, 127.3,127.2, 109.2, 107.7, 80.6, 77.7, 77.1, 76.9, 75.7, 72.0, 70.8, 49.4,36.9, 31.9, 29.7, 29.5, 29.3, 29.2, 28.4, 27.0, 26.6, 26.4, 26.3, 25.5,25.4, 22.6, 19.3, 14.1. MALDI-MS (positive mode, DHB): m/z 1217.7[M+Na]⁺. Anal. Calcd for C₇₆H₁₂₇NO₇Si (1194. 90): C, 76.39; H, 10.71; N,1.17. Found: C, 76.41; H, 10.75; N, 1.22.

(c) The procedures of 14(c) above was repeated but using the product of(b) above to give

which was purified by flash chromatography (3:7 ethyl acetate/petroleumether), the compound was obtained as a colorless solid, in a yield of85%. R_(f)-0.40 (4:6, ethyl acetate:petroleum ether). [α]_(D) ²⁵=−3.1 (c0.5, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 7.38-7.36 (m, 5H), 5.58 (d,J=9.0 Hz, 1H), 4.69 (d, J=5.2 Hz, 1H), 4.13-3.91 (m, 5H), 3.60 (dd,J=9.7, 3.2 Hz, 1H), 3.33 (dd, J=9.7, 2.5 Hz, 1H), 3.21 (dd, J=10.5, 5.2Hz, 1H), 3.10 (dd, J=10.5, 3.5 Hz, 1H), 2.12 (dt, =7.5, 3.2 Hz, 2H),1.58-1.24 (m, 72H), 1.42 (s, 6H), 1.39 (s, 3H), 1.31 (s, 3H), 0.87 (t,J=6.5 Hz, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.4, 139.6, 128.5, 128.3,126.9, 109.9, 107.8, 81.9, 77.7, 77.2, 76.8, 75.9, 74.9, 71.7, 71.1,48.1, 36.9, 31.9, 29.7, 29.5, 29.3, 28.9, 28.0, 27.3, 27.2, 26.4, 25.8,25.7, 22.6, 14.1. MALDI-MS (positive mode, CHCA): m/z 979.5 [M+Na]⁺.Anal. Calcd for C₆₀H₁₀₉NO₇ (956.51): C, 75.34; H, 11.49; N, 1.46. Found:C, 75.43; H, 11.48; N, 1.44.

(d) The procedures of 14(d) above was repeated but using the product of(c) above to give

as a colorless solid, hereafter referred to as Compound 15(‘4-(R)-Phenyl Threitol Ceramide’) obtained at a yield of 60%. ¹H NMR(600 MHz, C₅D₅N): δ 8.46 (d, J=8.5 Hz, 1H), 7.82-7.28 (m, 5H), 5.46 (d,J=5.4 Hz, 1H), 5.14-511 (m. 1H), 4.25-4.00 (m, 8H), 2.38 (m, 2H),1.88-1.25 (m, 72H), 0.86 (t, J=6.5 Hz, 6H). ¹³C NMR (150.9 MHz, C₅D₅N):δ 173.3, 144.4, 128.4, 127.7, 127.3, 76.6, 76.2, 75.3, 74.4, 72.7, 71.2,71.0, 51.7, 36.8, 33.8, 32.1, 30.3, 30.1, 30.0, 29.8, 29.7, 29.6, 26.6,26.3, 22.9, 14.2. MALDI-MS (positive mode, CHCA): m/z 899.5 [M+Na]⁺.Anal. Calcd for C₅₄H₁₀₁NO₇ (876.38): C, 74.01; H, 11.62; N, 1.60. Found:C, 74.05; H, 11.68; N, 1.65.Compound 16: 4-(S)-Phenyl Threitol-22-(Z)-Ceramide

(a) The procedure of 1(b) above was repeated using 22-(Z)-hexacosenoicacid with the product of 9(b) above e.g.

to give

which was purified by flash chromatography (5:9.5, ethylacetate:toluene) as a colourless solid (yield: 72%). R_(f)=0.53 (5:9.5,ethyl acetate:toluene). [α]_(D) ²⁵=+24.6 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.70-7.15 (m, 15H), 5.65 (d, J=8.5 Hz, 1H), 5.36-5.31 (m,2H), 4.83 (d, J=4.5 Hz, 1H), 4.17-4.06 (m, 4H), 3.84 (dd, J=7.5, 4.5 Hz,1H), 3.58 (dd, J=9.2, 2.2 Hz, 1H), 3.38 (dd, J=9.7, 2.1 Hz, 1H), 3.16(br, d, J=4.5 Hz, 2H), 2.13-1.94 (m, 6H), 1.61-1.17 (m, 76H), 1.04 (s,9H), 0.91-0.83 (m, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.2, 140.3-127.0(m, 17C), 109.2, 107.6, 81.5, 77.7, 76.8, 75.8, 75.6, 73.0, 70.9, 60.2,48.1, 36.8, 31.8, 29.7, 29.6, 29.5, 29.4, 29.35, 29.31, 29.2, 28.9,28.0, 27.15, 27.11, 27.0, 26.8, 26.4, 25.7, 25.6, 22.8, 22.6, 14.1,13.8. MALDI-MS (positive mode, DHB): m/z 1214.3 [M+Na]⁺.

(b) The procedure of 14(c) above was repeated using the product of (a)above and purified by flash chromatography (4:6 ethyl acetate:petroleumether) to give

as colourless solid (yield: 98%). R_(f)=0.31 (3:7, ethylacetate:petroleum ether). [α]_(D) ²⁵=+14.9 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.40-7.26 (m, 5H), 5.66 (d, J=9.0 Hz, 1H), 5.37-5.33 (m,2H), 4.88 (dd, J=5.5, 2.7 Hz, 1H), 4.23-3.96 (m, 5H), 3.60 (dd, J=10.0,4.0 Hz, 1H), 3.39 (dd, J=9.5, 2.2 Hz, 1H), 3.23 (dd, J=10.5, 5.2 Hz,1H), 3.13 (dd, J=10.5, 4.0 Hz, 1H), 3.09 (d, J=2.7 Hz, 1H), 2.12 (dt,J=10.5, 7.5, 3.5 Hz, 2H), 2.05-1.96 (m, 4H), 1.60-1.25 (m, 76H),0.92-0.85 (m, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.4, 139.6, 130.0,129.5, 128.2, 126.1, 109.2, 107.8, 80.9, 77.6, 76.6, 76.0, 73.0, 72.0,71.1, 48.1, 36.8, 31.8, 29.7, 29.67, 29.60, 29.5, 29.38, 29.33, 29.27,29.24, 28.8, 27.9, 27.1, 27.0, 26.9, 26.4, 25.69, 25.67, 22.8, 22.6,14.0, 13.7. MALDI-MS (positive mode, DHB): m/z 976.7 [M+Na]⁺.

(c) The procedure of 14(d) above was repeated using the product of (b)above to obtain a colourless solid

hereafter referred to as Compound 16 (‘4-(S)-PhenylThreitol-22-(Z)-Ceramide’), (yield: 67%). ¹H NMR (250 MHz, C₅D₅N): δ8.43 (d, J=8.7 Hz, 1H), 7.70-7.16 (m, 5H), 5.39-5.28 (m, 3H), 5.08-5.02(m, 1H), 4.89-4.79 (m, 1H), 4.23-4.07 (m, 5H), 4.05-3.95 (m, 2H), 2.30(br. t, J=7.5 Hz, 2H), 1.99-1.87 (m, 4H), 1.80-1.67 (m, 4H), 1.17-1.13(m, 60H), 0.78-0.73 (m, 6H). MALDI-MS (positive mode, DHB): m/z 897.1[M+Na]⁺. Anal. Calcd for C₅₄H₉₉NO₇ (873.74): C, 74.18; H, 11.41; N,1.60. Found: C, 74.16; H, 11.49; N, 1.68.Compound 17: 4-(R)-Phenyl Threitol-22-(Z)-Ceramide

(a) The procedure of 1(b) was repeated using 22-(Z)-hexacosenoic acidwith the product of 15(b) above e.g.

to give

which was purified by flash chromatography (5:9.5, ethylacetate:toluene) as a colourless solid (yield: 75%). R_(f)=0.57 (5:9.5,ethyl acetate:toluene). [α]_(D) ²⁵=−6.0 (c 1.0, CHCl₃). ¹H NMR (250 MHz,CDCl₃): δ 7.75-7.18 (m, 15H), 5.68 (d, J=8.0 Hz, 1H), 5.40-5.33 (m, 2H),4.72 (d, J=6.5 Hz, 1H), 4.12-4.00 (m, 3H), 3.93 (dd, J=8.0, 6.5 Hz, 1H),3.79-3.72 (m, 1H), 3.53 (br. d, J=9.5 Hz, 1H), 3.24 (br. d, J=9.5 Hz,1H), 3.02-2.91 (m, 2H), 2.16-1.96 (m, 6H), 1.60-1.25 (m, 76H), 1.04 (s,9H), 0.92-0.85 (, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.3, 139.6-127.2(m, 17C), 109.1, 107.7, 80.6, 77.7, 77.1, 76.9, 75.7, 72.1, 70.8, 48.1,36.9, 31.9, 29.74, 29.70 29.6, 29.5, 29.39, 29.35, 29.30, 29.2, 29.0,28.0, 27.2, 26.9, 26.7, 26.4, 25.8, 25.7, 22.8, 22.6, 14.1, 13.7.MALDI-MS (positive mode, DHB): m/z 1214.4 [M+Na]⁺.

(b) The procedure of 15(c) was repeated using the product of (a) aboveand purified by flash chromatography (4:6 ethyl acetate:petroleum ether)to give pure

as a colourless solid (yield: 98%). R_(f)=0.32 (3:7, ethylacetate:petroleum ether). [α]_(D) ²⁵=+5.5 (c 1.0, CHCl₃). ¹H NMR (250MHz, CDCl₃): δ 7.39-7.30 (m, 5H), 5.56 (d, J=9.2 Hz, 1H), 5.37-5.32 (m,2H), 4.68 (t, J=4.5 Hz, 1H), 4.13-3.94 (m, 5H), 3.59 (dd, J=9.5, 3.5 Hz,1H), 3.32 (dd, J=9.5, 3.5 Hz, 1H), 3.20 (dd, J=10.5, 5.2 Hz, 1H), 3.10(dd, J=10.2, 3.8 Hz, 1H), 2.92 (d, J=4.5 Hz, 1H), 2.11 (dt, =10.5, 7.5,3.5 Hz, 2H), 2.03-1.95 (m, 4H), 1.60-1.24 (m, 76H), 0.91-0.84 (m, 6H).¹³C NMR (62.5 MHz, CDCl₃): δ 172.3, 139.6, 130.0, 129.5, 128.4, 126.9,109.8, 107.7, 81.8, 77.6, 76.7, 75.8, 74.9, 71.7, 71.1, 48.0, 36.8,31.8, 29.7, 29.69, 29.60, 29.5, 29 38, 29.32, 29.28, 29.27, 29.24, 28.9,27.9, 27.29, 27.22, 27.1, 26.4, 25.7, 25.6, 22.8, 22.6, 14.0, 13.7.MALDI-MS (positive mode, DHB): m/z 976.6 [M+Na]⁺.

(c) The procedure of 15(d) was repeated using the product of (b) aboveto obtain a colourless solid

hereafter referred to as Compound 17 (‘4-(R)-PhenylThreitol-22-(Z)-Ceramide’), (yield: 63%). ¹H NMR (250 MHz, C₅D₅N): δ8.42 (d, J=8.7 Hz, 1H), 7.73-7.16 (m, 5H), 5.38-5.30 (m, 3H), 5.05-5.00(m, 1H), 4.19-3.98 (m, 7H), 3.92-3.86 (m, 1H), 2.28 (br. t, J=7.5 Hz,2H), 1.98-1.87 (m, 4H), 1.80-1.65 (m, 4H), 1.17-1.12 (m, 60H), 0.78-0.71(m, 6H). MALDI-MS (positive mode, DHB): m/z 897.4 [M+Na]⁺. Anal. Calcdfor C₅₄H₉₉NO₇ (873.74): C, 74.18; H, 11.41; N, 1.60. Found: C, 74.11; H,11.51; N, 1.66.

Synthesis of (19Z,22Z)-Hexacosadienoic Acid

(a) The procedure for ‘Synthesis 22-(Z)-Hexacosanoic acid’, step (a)above was repeated from 11-bromoundecanoic acid to give

in 97% yield, ¹H NMR (250 MHz, CDCl₃): δ 4.58 (t, J=3.2 Hz, 1H),3.92-3.83 (m, 1H), 3.78-3.72 (m, 1H), 3.65 (s, 3H), 3.52-3.46 (m, 1H),3.42-3.36 (m, 1H), 2.30 (t, J=7.5 Hz, 2H), 1.88-1.15 (m, 10H), 1.25 s,28H). ¹³C NMR (62.5 MHz, CDCl₃): δ 174.2, 98.7, 67.6, 62.2, 51.3, 34.0,30.7, 29.7, 29.63, 29.60, 29.5, 29.45, 29.40, 29.2, 29.1, 26.2, 25.4,24.9, 19.6. MALDI-MS (positive mode, CHCA): m/z 436.1 [M+Na]⁺.

(b) The procedure from the ‘Synthesis 22-(Z)-Hexacosanoic acid’, step(b) above, was repeat for (a) above to give

in 98% yield, ¹H NMR (250 MHz, CDCl₃): δ 3.67 (s, 3H), 3.63 (t, J=6.5Hz, 2H), 2.30 (t, J=7.5 Hz, 1H), 1.64-4.51 (m, 4H), 1.25 (br. s, 26H).¹³C NMR (62.5 MHz, CDCl₃): δ 174.3, 63.0, 51.4, 34.0, 32.7, 29.63,29.60, 29.5, 29.58, 29.56, 29.4, 29.2, 29.1, 25.7, 24.9. MALDI-MS(positive mode, CHCA): m/z 351.9 [M+Na]⁺.

(c) The procedure from the ‘Synthesis 22-(Z)-Hexacosanoic acid’, step(c) above, was repeat for (b) above to give

in 90% yield, ¹H NMR (250 MHz, CDCl₃): δ 9.76 (s, 1H), 3.66 (s, 3H),2.39 (dt, J=14.0, 7.5, 2.0 Hz, 2H), 2.26 (t, J=7.5 Hz, 2H), 1.66-1.56(m, 4H), 1.25 (br. s, 26H). ¹³C NMR (62.5 MHz, CDCl₃): δ 202.6, 173.4,50.7, 43.4, 315, 29.29, 29.23, 29.08, 29.01, 28.9, 28.79, 28.76, 24.5,21.6. ESI-MS (positive mode) (326.2): 349.3 [M+Na]⁺.

(d) The procedure from the ‘Synthesis 22-(Z)-Hexacosanoic acid’, step(d) above, was repeat for (c) above to give

in 98% yield, ¹H NMR (250 MHz, CDCl₃): δ 5.37-5.34 (m, 4H), 3.66 (s,3H), 2.78 (t, J=6.2 Hz, 2H), 2.30 (t, J=7.5 Hz, 2H), 2.08-1.99 (m. 4H),1.64-1.58 (m, 2H), 1.42-1.25 (m, 30H), 0.91 (t, 7.5 Hz, 31-1). ¹³C NMR(62.5 MHz, CDCl₃): δ 174.1, 130.0, 129.7, 128.0, 127.8, 51.2, 34.0,29.64, 29.60, 29.55, 29.51, 29.4, 29.27, 29.22, 29.1, 27.1, 25.5, 24.8,22.7, 13.7.

(e) The product of (d) above (1.23 g, 3.022 mmol) in THF (10 mL) and 2 Npotassium hydroxide (12 mL) was heated to reflux for 8 h, and acidifiedwith 2 N hydrochloric acid (˜pH 1-2). Extracted with diethyl ether (2×30mL), dried and concentrated to give colorless solid which wasrecrystallized from glacial acetic acid to give colorless crystals(yield: 52%), and mother liquid was concentrated and purified by flashchromatography to give

(19Z,22Z)-Hexacosadienoic acid as colorless crystals in 42% yield, ¹HNMR (250 MHz, CDCl₃): δ 5.37-3.32 (m, 4H), 2.76 (t, J=6.2 Hz, 2H), 2.33(t, J=7.5 Hz, 2H), 2.06-1.98 (m, 4H), 1.67-1.55 (m, 2H), 1.32-1.23 (m,30H), 0.90 (t, J=7.5 Hz, 3H). ¹³C NMR. (62.5 MHz, CDCl₃): δ 180.1,130.1, 129.8, 128.1, 127. 9, 34.0, 29.6, 29.59, 29.55, 29.4, 29.3, 29.2,29.0, 27.2, 25.6, 24.6, 22.8, 13.8. BSI-MS (negative mode) (392.3):391.4 [M-H]⁻.Compound 18: Threitol-(19Z,22Z)-Ceramide

(a) The procedure of 1(b) above was repeated using with(19Z,22Z)-Hexacosadienoic acid and the product of 3(b) e.g.

to give

colourless liquid, which is solidifies upon standing. (yield: 71%).R_(f)=0.24 (3:7, ethyl acetate:petroleum ether). [α]_(D) ²⁵=+13.6 (c1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃): δ 5.73 (d, J=9.5 Hz, 1H),5.43-5.33 (m, 4H), 4.24-4.15 (m, 1H), 4.08-3.99 (m, 3H), 3.92-3.86 (m,1H), 3.80-3.65 (m, 5H), 3.60-3.54 (m, 2H), 2.77 (t, J=6.2 Hz, 2H), 2.14(dt, J=10.5, 7.5, 3.0 Hz, 2H), 2.07-1.99 (m, 4H), 1.61-1.24 (m, 74H),0.93-0.84 (m, 6H). ¹³C NMR (62.5 MHz, CDCl₃): δ 172.5, 130.0, 129.7,128.0, 127.8, 109.2, 107.9, 79.1, 77.6, 76.6, 76.1, 71.8, 71.6, 62.3,48.1, 36.8, 31.8, 29.6, 29.5, 29.4, 29.36, 29.30, 29.26, 29.21, 28.9,27.8, 27.1, 26.9, 26.4, 25.67, 25.63, 25.5, 22.7, 22.6, 14.0, 13.7.MALDI-MS (positive mode, DHB): m/z 899.1 [M+Na]⁺.

(b) The procedure of 1(c) above was followed using the product of (a)above to give

colourless solid (yield: 67%), hereafter referred to as Compound 18(‘Threitol-(19Z, 22Z)-Ceramide’). ¹H NMR (250 MHz, C₅D₅N): δ 8.65 (d,J=8.5 Hz, 1H), 5.66-5.55 (m, 4H), 5.30-5.24 (m, 1H), 4.63-4.45 (m, 1H),4.42-4.32 (m, 9H), 4.18 (d, J=5.5 Hz, 2H), 3.01 (t, J=6.2 Hz, 2H), 2.54(t, J=7.5 Hz, 2H), 2.28-2.14 (m, 4H), 2.03-1.87 (m, 4H), 1.48-1.36 (m,58H), 1.02-0.97 (m, 6H). MALDI-MS (positive mode, Matrix CHCA): m/z819.8 [M+Na]⁺.

1. A composition useful in stimulating a T cell response comprising acompound of formula:

and an immunostimulatory molecule in which R¹ represents a hydrophobicmoiety adapted to occupy the C′ channel of human CD1d, R² represents ahydrophobic moiety adapted to occupy the A′ channel of human CD1d, suchthat R¹ fills at least at least 30% of the occupied volume of the C′channel compared to the volume occupied by the terminal nC₁₄H₂₉ of thesphingosine chain of α-galactosylceramide when bound to human CD1d andR² fills at least 30% of the occupied volume of the A′ channel comparedto the volume occupied by the terminal nC₂₅H₅₁ of the acyl chain ofα-galactosylceramide when bound to human CD1d R³ represents hydrogen orOH, R^(a) and R^(b) each represent hydrogen and in addition, when R³represents hydrogen, R^(a) and R^(b) together may form a single bond, Xrepresents or —CHA(CHOH)_(n)Y or —P(═O)(O⁻)OCH₂(CHOH)_(m)Y, in which Yrepresents CHB₁B₂, n represents an integer from 1 to 4, m represents 0or 1, A represents hydrogen, one of B₁ and B₂ represents H, OH orphenyl, and the other represents hydrogen or one of B₁ and B₂ representshydroxyl and the other represents phenyl, in addition, when n represents4, then A together with one of B₁ and B₂ together forms a single bondand the other of B₁ and B₂ represents H, OH or OSO₃H andpharmaceutically acceptable salts thereof.
 2. The composition of claim1, wherein said T cell response is a CTL response.
 3. The composition ofclaim 1, wherein said immunostimulatory molecule is a protein, peptide,or nucleic acid molecule.
 4. The composition of claim 3, wherein saidprotein or peptide is a tumor antigen.
 5. The composition of claim 4,wherein said tumor antigen is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1,SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 or CT-7.
 6. Amethod for inducing a T cell response in a subject in need thereof,comprising administering an amount of the composition of claim 1 to saidsubject in an amount sufficient to induce said T cell response.
 7. Amethod for inducing a T cell response in a subject in need thereof,comprising administering an amount of the composition of claim 5 to saidsubject in an amount sufficient to induce said T cell response.